Cell non-Hodgkin Lymphoma Research Articles

Divergent Oxidative Stress in Normal Tissues and Inflammatory Cells in Hodgkin and Non-Hodgkin Lymphoma.

Previous studies reported mitochondrial and endoplasmic reticulum redox stress in peripheral blood mononucleated cells (PBMCs) of treatment-naïve Hodgkin lymphoma (HL) patients. Here, we assessed whether this response also applies to non-HL (NHL) patients, and whether the oxidative damage is a selective feature of PBMCs or, rather, also affects tissues not directly involved in the inflammatory response. Isolated PBMCs of 28 HL, 9 diffuse large B cell lymphoma, 8 less aggressive-NHL, and 45 controls underwent flow cytometry to evaluate redox stress and uptake of the glucose analogue 2-NBDG. This analysis was complemented with the assay of malondialdehyde (MDA) levels and enzymatic activity of glucose-6P-dehydrogenase and hexose-6P-dehydrogenase (H6PD). In all lymphoma patients, 18F-fluoro-deoxyglucose uptake was estimated in the myocardium and skeletal muscles. Mitochondrial reactive oxygen species generation and MDA levels were increased only in HL patients as well as H6PD activity and 2-NBDG uptake. Similarly, myocardial FDG retention was higher in HL than in other groups as opposed to a similar tracer uptake in the skeletal muscle. Redox stress of PBMCs is more pronounced in HL with respect to both NHL groups. This phenomenon is coherent with an increased activity of H6PD that also extends to the myocardium.

Role of Flow Cytometry in Diagnosis of Non-Hodgkin’s Lymphoma: A Study at RIMS, Ranchi, India

Purpose: Non-Hodgkin’s lymphomas (NHL) are cancers of mature B, T and NK cells. Diagnosis of NHL is based on immunophenotyping by immunohistochemistry of excised lymph node or flow cytometric immunophenotyping. FNA cytology with immunolabelled flow cytometry can in some circumstance serve as a replacement of open biopsy and conventional histology and immunohistochemistry. Method: The present study consisted of 52 patients suffering from lymphoreticular malignancies who attended and/or were admitted in the Rajendra Institute of Medical Sciences, Ranchi, during the period from March 2020 to August 2021. Results: Out of 54 cases suspicious of Lymphoreticular malignancy, 2 were found to be non-lymphoid origin by flow cytometry of fine needle aspirate and sample collection was inadequate in 3 cases (5.5%). On Immunophenotyping, 44 cases (88%) were found to be B-NHL and 5 cases (10%) were T-NHL. Furthermore, it was sublassified as DLBCL 16 cases (32.6%), FL 6 cases (12.2%), MZBCL 3 cases (6.1%), CLL/SLL 5 cases (10.2%), MCL 2 cases (4.08%), Unclassified B-NHL 12 cases (20.3%) and Mature T cell NHL 5 cases (10.2%). Conclusion: Flowcytometry can be a great tool in diagnosis and classification of NHL and can be used alternatively to conventional excisional Lymph node biopsy and immunohisto-chemistry. Keywords: Flow cytometry, immunophenotyping, Non Hodgkin’s Lymphoma, FNA cytology

FREQUENCY OF CAUSES OF PANCYTOPENIA IN BONE MARROW SAMPLES OF PATIENTS

Objective: The aim of this study is to find out the common causes that lead to pancytopenia in our population specially in the province of Punjab. Material and Methods: This is a cross sectional study that was conducted at Chughtai Institute of Pathology, Lahore. The duration of the study was from June 2020 to December 2021. A total of 210 patients were included in the study. A through history was taken and complete physical examination was done prior to the bone marrow biopsy procedure. Peripheral blood sample was run on Sysmex XN 9000 automated hematology analyzer. Bone marrow aspirate and trephine biopsy were thoroughly examined and immunohistochemical stains were applied where required. The data was analyzed using SPSS 23.0 and frequencies were calculated. Results: Most common causes of pancytopenia in our study were megaloblastic anemia (18.1%) followed by aplastic anemia (12.9%), immune mediated causes (12.4%), acute myeloid leukemia (11%), hypocellular marrow (9%) myelodysplastic syndrome (6.2%), hypersplenism (5.2%) and Precursor B acute lymphoblastic leukemia (5.2%) (Fig. 2). Other causes include B cell non-Hodgkin lymphoma (4.8%), mixed deficiency anemia (4.2%), hypersplenism (3.8%), Gaucher’s disease (1.4%), infections (1%) and other miscellaneous causes (4.8%). Conclusion: The study conducted at our institute showed that majority of patients had benign causes (69.2%) leading to pancytopenia, the most common cause being megaloblastic anemia. Malignant causes constitute only a minority of cases of pancytopenia. Key Words: Bone marrow biopsy, Frequency, Pancytopenia

Chimeric antigen receptor T-cells: Properties, production, and quality control.

Chimeric antigen receptor (CAR) T-cell therapy is a novel adoptive T-cell immunotherapy for haematological malignancies. First introduced into clinical practice in 2017, CAR T-cell therapy is now finding its place in the management of lymphoid malignancies, primarily of B-cell lineage, including lymphoblastic leukaemia, non-Hodgkin lymphoma and plasma cell myeloma, with remarkable therapeutic outcomes. CAR T-cells are a customised therapeutic product for each patient. Manufacture commences with collection of autologous T-cells, which are then genetically engineered ex vivo to express transmembrane CARs. These chimeric proteins consist of an antibody-like extracellular antigen-binding domain, to recognise specific antigens on the surface of tumour cells (e.g. CD19), linked to the intracellular co-stimulatory signalling domains of a T-cell receptor (e.g. CD137). The latter is required for in vivo CAR T-cell proliferation, survival, and durable efficacy. Following reinfusion, CAR T-cells harness the cytotoxic capacity of a patient's immune system. They overcome major mechanisms of tumour immuno-evasion and have potential to generate robust cytotoxic anti-tumour responses. This review discusses the background to CAR T-cell therapies, including their molecular design, mechanisms of action, methods of production, clinical applications and established and emerging technologies for CAR T-cell evaluation. It highlights the need for standardisation, quality control and monitoring of CAR T-cell therapies, to ensure their safety and efficacy in clinical management.

Validating the anti-lymphoma pharmacodynamic actions of the endocannabinoids on canine non-Hodgkin lymphoma

AimsThis study established the in vitro anti-lymphoma pharmacodynamic actions of the endocannabinoids (anandamide—AEA and 2-arachidonoylglycerol—2AG) on canine non-Hodgkin lymphoma (NHL) and human NHL cells. Main methodsThe expression of cannabinoid (CB1 and CB2) receptors in various canine NHL cells {1771, CLBL-1, CLL-1, peripheral blood mononuclear cells (PBMCs)} was studied using Quantitative real-time PCR (RT-qPCR). Anti-lymphoma cell viability assay was performed to assess the effect of endocannabinoids on various canine and human NHL cells (1771, CLBL-1, CLL-1, Ramos cells). The spectrophotometric and fluorometric procedures evaluated oxidative stress, inflammation, apoptosis, and mitochondrial function markers. SAS® and Prism-V La Jolla, CA, USA, were used for statistical analysis. Key findingsThe current study validated the presence of CB1 and CB2 receptors in the canine NHL cells. There was a significantly higher expression of CB1 and CB2 receptors in B-cell lymphoma (BCL) cells (1771, CLBL-1, Ramos) compared to canine T-cell lymphoma (TCL) cells (CL-1). AEA and 2AG dose and time-dependently exhibited significant but differential anti-lymphoma effects on canine and human NHL cells. Anti-lymphoma pharmacodynamic actions of the endocannabinoids in the canine 1771 NHL cells revealed a significant alteration in the markers of oxidative stress, inflammation, and a decrease in mitochondrial function without altering the apoptotic markers. SignificanceEstablishing the anti-lymphoma pharmacodynamic actions of endocannabinoids may provide new therapeutic interventions and expedite cannabinoid research.

Elraglusib (formerly 9-ING-41) possesses potent anti-lymphoma properties which cannot be attributed to GSK3 inhibition

Elraglusib (formerly 9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3β (GSK3β) undergoing clinical trials for the treatment of various cancers including non-Hodgkin lymphoma (NHL). The drug reduces proliferation of several NHL cell lines and has efficacy in xenograft models of the disease. To confirm the importance of its action on GSK3β, we treated 3 lymphoma cell lines with selective, structurally distinct GSK3 inhibitors: CT99021, SB216763, LY2090314, tideglusib, and elraglusib. Stabilization of β-catenin and reduced phosphorylation of CRMP2, two validated targets of GSK3, were used as functional read-outs for GSK3 inhibition. CT99021, SB216763, and LY2090314 failed to reduce proliferation or viability in any cell line at concentrations that stabilized β-catenin and reduced CRMP2 phosphorylation. There was partial reduction of CRMP2 phosphorylation but no significant effect on β-catenin at cytotoxic doses of elraglusib. There was no indication of GSK3 inhibition at doses of tideglusib that affected cell viability and apoptosis. Cell-free kinase screening confirmed several other targets of elraglusib, distinct from the GSK3 inhibitors with no anti-lymphoma actions, including PIM kinases and MST2. These data question GSK3 as the target of elraglusib in lymphoma, and hence the utility of GSK3 expression as a ‘stand-alone’, therapeutic biomarker in NHL.7CsbmemxsJeEQuU9mL3kzMVideo

Artesunate synergistically promotes sorafenib‑induced apoptosis and ferroptosis in non‑Hodgkin lymphoma cells through inhibition of the STAT3 pathway.

Despite the development of advanced therapies, the prognosis of non‑Hodgkin lymphoma (NHL) remains unsatisfactory due to refractory and relapsed cases. Artesunate (ART) and sorafenib (SOR) both exert potential antitumor activity in lymphoma. The present study aimed to investigate whether ART and SOR produce synergistic anti‑lymphoma effects, and to determine the potential underlying mechanisms. Cell viability assay, flow cytometry, malondialdehyde assay, GSH assay and western blotting were performed to evaluate cell viability, and changes in apoptosis, autophagic vacuoles, reactive oxygen species, mitochondrial membrane potential, lipid peroxidation and protein expression. The results demonstrated that ART and SOR synergistically inhibited the viability of NHL cells. ART and SOR also synergistically induced apoptosis, and markedly increased the expression levels of cleaved caspase‑3 and poly (ADP‑ribose) polymerase. Mechanistically, ART and SOR synergistically induced autophagy, and rapamycin enhanced the ART‑ or SOR‑induced inhibition of cell viability. Furthermore, it was demonstrated that ferroptosis promoted ART‑ and SOR‑induced cell death through increasing lipid peroxides. Erastin enhanced the inhibitory effects of ART and SOR on cell viability, whereas ferrostatin‑1 reduced the ART‑ and SOR‑induced apoptosis of SU‑DHL4 cells. Further studies revealed that signal transducer and activator of transcription3 (STAT3) contributed to ferroptosis induced by ART and SOR in NHL cells, and genetic inhibition of STAT3 promoted ART/SOR‑induced ferroptosis and apoptosis, concomitantly reducing the expression levels of glutathione peroxidase4 and myeloid cell leukemia‑1. Moreover, the combined treatment of ART and SOR exerted inhibitory effects on tumor growth, as well as antiangiogenic activity, resulting in the inhibition of CD31 expression in a xenograft model. Collectively, these findings indicated that ART acted synergistically with SOR to inhibit cell viability, and to induce apoptosis and ferroptosis through regulating the STAT3 pathway in NHL. Notably, ART and SOR may act as potential therapeutic agents for the treatment of lymphoma.

LP-284, a small molecule acylfulvene, exerts potent antitumor activity in preclinical non-Hodgkin's lymphoma models and in cells deficient in DNA damage repair

Despite advances in therapies treating non-Hodgkin's lymphoma (NHL), 20~40% of patients experience relapsed or refractory disease. While solid tumors with homologous recombination deficiencies have been successfully targeted with synthetic lethal agents such as poly-ADP ribose polymerase (PARP) inhibitors, such synthetic lethality strategy has not yet been approved to treat patients with NHL. Here we investigated the mechanism of action (MoA) and therapeutic potential of a new-generation acylfulvene compound, LP-284, in both in vitro and in vivo NHL models. One of LP-284's MoA includes inducing the repair of double-strand DNA break (DSB). We found that LP-284 exerts nanomolar potency in a panel of hematological cancer cell lines including fifteen NHL cell lines. In vivo, LP-284 treatment prolongs the survival of mantle cell lymphoma (MCL) cell line JeKo-1 derived xenograft mice by two-fold and shows increased efficacy over bortezomib and ibrutinib. In addition, LP-284 is capable of inhibiting tumor growth of JeKo-1 xenografts that are refractory to bortezomib or ibrutinib. We further showed that LP-284 is particularly lethal in cells with deficient DNA damage response and repair, a targetable vulnerability in NHL.

Impact of neighborhood deprivation on disability free survival in patients with aggressive B cell non-Hodgkin lymphoma.

7564 Background: Advancements in treatment over the last decade have greatly improved survival in B cell non-Hodgkin’s lymphoma (NHL), yet not all patients (pts) benefit the same. Non-Hispanic Black (NHB), uninsured and lower socioeconomic status pts continue to suffer worse survival. However, the impact of neighborhood deprivation on disability has not been explored. We aimed to assess the association between neighborhood deprivation and disability free survival (DFS) in NHL pts. Methods: 258 aggressive NHL pts treated between 2005-2020 were included. Pts without documented follow up were excluded. Race/ethnicity was categorized as non-Hispanic white (NHW), Hispanic, NHB and other. Primary outcomes included progression free survival (PFS), overall survival (OS) and DFS. DFS was defined as time to needing any rehabilitative, home-health or skilled nursing services immediately following induction therapy, indicating disability and lack of functional independence. Neighborhood deprivation was assigned using the national Area Deprivation Index (ADI), a validated measure classifying neighborhood adversity. Pts were categorized into high (67-99), intermediate (34-66) or low (1-33) ADI scores, with least disadvantaged designated by lower scores. Kaplan-Meier analyses were used to compare PFS, OS and DFS. Results: Demographic data is shown in the table; 45% were female, 65% were NHW, 10% were NHB, 8% were Hispanic and 10% were other. Median age at diagnosis was 57 years. There was no difference in PFS or OS between racial/ethnic groups (p=0.91 and p=0.23 respectively). Payor source influenced both PFS and OS (p<0.001 and p<0.001 respectively), with self-pay associated with worse PFS and OS. Neighborhood deprivation based on ADI did not influence PFS (p=0.33) or OS (p=0.76). High ADI score was associated with worse DFS compared to lower ADI scores (p=0.03). ADI score differed significantly based on race/ethnicity (p<0.0001), with more NHB pts residing in areas of high ADI scores (40% vs 5% NHW vs 10% Hispanic). There was no association between ADI score and a pt’s ECOG performance status, IPI score or albumin at diagnosis (p=0.87, p=0.78 and p=0.59 respectively). Conclusions: NHL pts with higher neighborhood adversity experience more disability after treatment despite similar survival. Further work is needed to better understand how neighborhood factors are contributing to disability before community-level strategies for improvement can be developed. [Table: see text]

MYC/BCL6 DOUBLE HIT LYMPHOMA NEGATIVE FOR T(3;8) BCL6::MYC FUSION IS ASSOCIATED WITH INFERIOR SURVIVAL, IN CONTRAST WITH T(3;8) POSITIVE PSEUDO‐DOUBLE HIT LYMPHOMA

Introduction: A small proportion of large B cell non-Hodgkin lymphoma (NHL) has MYC and BCL6 rearrangements, detectable by fluorescence in-situ hybridisation (FISH) break-apart probes. There are conflicting reports on the prognosis of this group. A proportion of them have a single t(3;8) BCL6::MYCtranslocation which accounts for both break-apart probe results. The prognostic significance of this t(3;8) positive group is unknown. Methods: We conducted a retrospective cohort study of cases of high grade B NHL, excluding Burkitt lymphoma, referred to a regional histopathology and FISH laboratory from 14 hospitals in the West Midlands, UK. Cases of MYC/BCL2 double-hit lymphoma without BCL6 rearrangement were excluded. Survival was analysed using univariate and multiple regression with Cox proportional hazards. Results: Data were collected for 106 cases of high grade B cell lymphoma with MYC rearrangements. BCL6 rearrangement by FISH break-apart probe (BCL6r) was seen in 61, of which 19/38 tested with a t(3;8) fusion probe were positive. The four groups, MYC single hit, BCL6r t(3;8) positive, BCL6r t(3;8) negative, and BCL6r t(3;8) untested, were well matched for baseline histological and clinical characteristics. Univariate survival analysis showed that the BCL6r t(3;8) negative group had inferior overall survival (OS) and progression-free survival relative to MYC single hit (hazard ratio (HR) 2.6 for death, p = 0.01), and the association was maintained in a multivariate analysis (HR 3.1, p = 0.01). No significant effect on survival was associated with the BCL6r t(3;8) positive group. Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, pathology and classification of lymphomas No conflicts of interests pertinent to the abstract.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: There is currently no consensus on the role of upfront ASCT for patients with T cell Non-Hodgkin lymphomas (T-NHL), especially in patients who have achieved complete remission (CR1) following induction chemotherapy. To date, no randomized control trials (RCTs) exist and available data is conflicting, especially with regards to T-NHL subtypes that might benefit. A systematic review and meta-analysis of all comparative studies (randomized, non-randomized and observational) was hence performed to address this question. Methods: A comprehensive, systematic search (from 1/2000 to 2/2022) of MEDLINE/PubMed, EMBASE and Cochrane databases was performed. PRISMA and Meta-Analysis Of Observational Studies in Epidemiology (MOOSE) guidelines were followed. Studies were screened based on predefined inclusion/exclusion criteria, and were critically appraised for outcomes of interest [progression free survival (PFS) and overall survival (OS)]. Quality of studies was assessed using Newcastle-Ottawa Scale. Hazard ratios (HRs) and corresponding 95% Cis were calculated, and the meta-analysis was performed using the random-effects model. Test for heterogeneity was performed using I2 statistic. Results: Of 3297 unique records, 17 studies (prospective = 6, retrospective = 11) were included. Median follow up in these studies ranged from 22 to 94 mths. Ten studies compared upfront ASCT versus No ASCT in patients with complete remission (CR1), while in 7 patients achieving either PR1 (first partial remission) or CR1 were included[MS1]. Of these, 9 studies included only transplant eligible patients. The T-NHL subtypes included in the studies were also heterogenous, with nodal T-NHLs, in particular Angioimmunoblastic T-NHL (AITL) being amongst the most common subtypes. Results: from the meta-analysis showed that PFS in T-NHL patients who underwent ASCT benefited (HR 0.64, 95% CI: 0.46–0.89, I2 = 74%) compared to not undergoing ASCT; however similar benefit was not observed in OS (HR 0.67, 95% CI: 0.36–1.23, I2 = 89%). Sensitivity analyses including only studies with patients transplanted in CR1, and studies involving transplant eligible patients showed similar findings. In the studies that evaluated patients with the AITL subtype, ASCT showed a significant benefit in PFS and OS, compared to not undergoing ASCT (PFS = HR 0.19, 95% CI: 0.11–0.32, I2 20%; OS = HR 0.27, 95% CI: 0.09–0.81, I2 88 (Figure 1). Keyword: stem cell transplant No conflicts of interests pertinent to the abstract.

A phase 1 study of AT101, a novel anti-CD19 CAR-T cell therapy targeting a membrane-proximal epitope of CD19, in patients with relapsed or refractory B cell non-Hodgkin lymphoma.

7522 Background: All the FDA-approved CD19 CAR-T products are based on FMC63 scFv, which binds to the membrane-distal region of CD19. We developed a novel anti-CD19 antibody clone (1218) that binds to a membrane-proximal epitope of CD19, thereby not competing with FMC63. AT101 is an autologous CAR T cell transduced with a lentiviral vector, including the CD19-CAR with a humanized scFv of 1218, 4-1BB costimulatory, and CD3zeta domain. Methods: In this phase 1 trial, patients (n = 3 per dose level; up to n = 18 in total) are treated with AT101 in 3 dose-escalation cohorts based on a standard 3 + 3 design. Each patient received a single intravenous dose of AT101 at dose level (DL) 1 (0.2 x 106 cells/kg), DL2 (1.0 x 106 cells/kg), or DL3 (5.0 x 106 cells/kg). The primary objective is to determine the safety, the maximum tolerated dose (MTD), and the recommended phase 2 dose (RP2D) of AT101. The secondary objective is to evaluate the pharmacokinetics of AT101 and the preliminary efficacy, such as overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), event-free survival (EFS), and overall survival (OS). Key eligibility criteria include patients aged ≥19 with histologically confirmed relapsed or refractory B-cell non-Hodgkin lymphoma (NHL). Results: Fourteen patients were enrolled from March 2022 to December 2022, and nine were treated. The median age of treated patients was 61.6 years (ranged from 39 to 84) after a median of 4 prior lines of therapy (range 2-10). Their subtypes of NHL were as follows: diffuse large B cell lymphoma (n = 4), follicular lymphoma (n = 3), mantle cell lymphoma (n = 1), or marginal zone lymphoma (n = 1). The dosing of AT101 at DL1 and DL2 was completed. The dosing at DL3 is ongoing. Across cohorts 1 and 2, no grade 3 or higher cytokine release syndrome (CRS) was reported. Among the first three patients at DL1, one dose-limiting toxicity (DLT) of grade 4 neurotoxicity was observed but resolved in a week without sequelae. No other DLTs were observed in the additional three patients at the DL1 and three at the DL2 cohort. Another one at DL2 experienced grade 1 CRS with grade 1 neurotoxicity. Five patients experienced Grade ≥3 hematologic toxicities. An ORR is 66.7% (4/6) in cohort 1 and 100% (3/3) in cohort 2, including six complete responses (CR, 50.0% [3/6] in cohort 1 and 100% [3/3] in cohort 2). As of February 13, 2023, all six CRs are ongoing, including two patents exceeding six months after the treatments. Updated results will be presented at the meeting, including the cohort of DL3. Conclusions: In this phase I study, AT101 was well tolerated at the first two dose levels and showed promising efficacy in relapsed or refractory B-cell NHL patients. The majority of adverse effects were transient and manageable. The administrations of DL3 are currently ongoing, and updated results will be presented at the meeting. Clinical trial information: NCT05338931 .

Safety and efficacy of CRISPR-based non-viral PD1 locus specifically integrated anti-CD19 CAR-T cells in patients with relapsed or refractory Non-Hodgkin's lymphoma: a first-in-human phase I study.

Thus far, all approved chimeric antigen receptor (CAR)-T products are manufactured using modified viruses, which increases the risk of tumorigenesis, costs and production time. We aimed to evaluate the safety and efficacy of a kind of virus-free CAR-T cells (PD1-19bbz), in which an anti-CD19 CAR sequence is specifically integrated at the PD1 locus using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, in adults with relapsed/refractory (r/r) B cell non-Hodgkin's lymphoma (B-NHL). This single-arm phase I dose-escalation clinical trial evaluated PD1-19bbz in adult patients with r/r B-NHL from May 3rd 2020 to August 10th 2021. The patients were recruited and treated at the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China. Patients underwent leukapheresis and lymphodepleting chemotherapy before PD1-19bbz infusion. After the dose-escalation phase including three cohorts: 2×106/kg, 4×106/kg, 6×106/kg with three patients at each dose level, the optimal biological dose was determined to be 2×106/kg, which was then applied to an extended cohort of nine patients. The primary endpoint was the incidence of dose-limiting toxicities (DLT). The secondary endpoint was the response and survival. This trial was registered at www.clinicaltrials.gov as #NCT04213469. Twenty-one patients received PD1-19bbz infusion. Among all treated patients, 19 (90%) patients were diagnosed with stage III or IV disease. Meanwhile, 19 (90%) were stratified as intermediate risk or worse. Of note, four participants had >50% programmed death ligand-1 (PD-L1) expression in pre-treatment tumour sample, including two with extremely high levels (∼80%). There was no DLT identified. Fourteen patients had low-grade (1-2) cytokine release syndrome and two patients received tocilizumab. Four patients experienced immune effector cell-associated neurotoxicity syndrome of grade 1-2. The most common adverse events were hematologic toxicities, including anaemia (n=6), lymphocyte count decreased (n=19), neutrophil count decreased (n=17), white blood cell count decreased (n=10), and platelet count decreased (n=2). All patients had objective response and 18 patients reached complete response. At a median follow-up of 19.2 months, nine patients remained in remission, and the estimated median progression-free survival duration was 19.5 months (95% confidence interval: 9.9-infinity), with the median overall survival not reached. In this first-in-human study of non-viral specifically integrated CAR-T products, PD1-19bbz exhibited promising efficacy with a manageable toxicity profile. A phase I/II trial of PD1-19bbz in a larger patient cohort is underway. National Key R&D Program of China, National Natural Science Foundation of China, Key Project of Science and Technology Department of Zhejiang Province, Shanghai Zhangjiang National Independent Innovation Demonstration Area, Key Projects of Special Development Funds.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: We previously performed global miRNA expression profiling of cHL vs. non-Hodgkin lymphoma (NHL) cell lines and sorted germinal centre B-cells (GCB). In the group of 79 deregulated miRNAs (p < 0.05) we became particularly interested in the upregulated miR-23a-3p, a potential oncomiR, predicted to regulate the expression of TNFAIP3 and the downregulated miR-148a-3p, a potential tumor suppressor miRNA, predicted to regulate the expression of IL15. Therefore, we aimed to decipher the role of miR-23a-3p and miR-148a-3p in cHL pathogenesis. Methods: Expression analyses were performed with TaqMan Advanced miRNA Assays versus two miRNA controls. Laser capture microdissection was used for primary HRS collection. 1,000 (miRNA expression) and 2 × 200 (DNA methylation) HRS cells were collected per case. Bisulfite pyrosequencing assay was designed using the PyroMark Software and sequencing performed using the PyroMark Q24 sequencer. MiRNA-mRNAs interactions were validated with Dual-Glo Luciferase Assay using respective miRNA mimics. For overexpression pre-miRNAs were cloned into the pCDH-CMV-MCS-EF1α-GreenPuro vector and transductions were performed in triplicate. Cell proliferation was measured using CCK-8 in four replications in three independent reactions by the GloMax®96 Microplate reader. Results: We confirmed the overexpression of miR-23a-3p (p < 0.001) and downregulation of miR-148a-3p (p < 0.014) in microdissected HRS cells (n = 10) vs. GCB cells (n = 10). Moreover, we observed inverse correlation of miR-148a-3p expression with DNA methylation level of an adjacent CpG island (r = −0.72, p < 0.01; cHL n = 7, NHL n = 10). Elevated DNA methylation of this CpG island was detected also in 2/6 cHL primary cases. Next, we validated interactions between miR-23a-3p and miR-148a-3p and their target genes. A reduction by 34% (p < 0.001) and 40% (p < 0.01) of the luciferase signal was observed in consequence of the interaction of miR-23a-3p mimic—TNFAIP3 3’UTR and miR-148a-3p mimic—IL15 3’UTR, respectively. Moreover, the abundance of TNFAIP3 protein was reduced after overexpression of the miR-23a (coexpressed with miR-27a) in L428 (p = 0.0018) and GCB6-16 (p = 0.0083) cell lines, compared to empty vector transductions. Whereas overexpression of miR-148a in three cHL cell lines caused a 32% decrease (p < 0.05) in proliferation in the KM-H2 cell line compared to empty vector transductions. The research was funded by: This research was funded by the National Science Center, Poland grants: 2014/14/M/NZ2/00529 to M.G. and the 2019/32/T/NZ5/00441 Etiuda scholarship to JP. SH is supported by the German Research Foundation (DFG, HA6145/3-1). MG, ADz-K and NR received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952304. Keywords: Hodgkin lymphoma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

Correlation between Germinal Center Differentiation and Double/Triple Hit Score in the Classification of Diffuse Large B Cell Non-Hodgkin Lymphoma: A Clinicopathological Approach

Background: Diffuse large B cell lymphoma (DLBCL) is considered the most common type of non-Hodgkin lymphoma. The global incidence of DLBCL has been doubled in the past decades, highlighting the need for more effective treatment regimens. The curability of DLBCL is heavily influenced by a number of factors, such as the age, the international prognostic index (IPI) score, the molecular cell of origin (COO) subtype, and presence/ absence of specific chromosomal rearrangements or protein expression. Recent gene profiling studies have classified DLBCL into two main categories, germinal center B-cell type (GCB) and activated B-cell type (ABC). Validation of these subtypes has become more applicable after the immunohistochemical algorithm suggested by Hans, which included three commercially available markers, CD10, BCL6, and MUM-1/IRF4. In addition to this classification, DLBCL prognostic models based on different sets of genes or immunohistochemical markers have been proposed. More recently, DLBCLs with translocations of MYC, along with a B-cell lymphoma 2 (BCL2) and/or B-cell lymphoma 6 (BCL6) rearrangement, are now called double-hit lymphoma (DHL) or triple-hit lymphoma (THL), respectively. Furthermore, the co-expre sion of MYC and BCL2 proteins without underlying rearrangements is considered a new adverse prognostic indicator termed double-expressor lymphoma (DEL). The present study is aiming to combine morphological, immunophenotypical, and genetic features of DLBCLs to reach a more reproducible subtyping. Aim: To identify the relation between clinicopathological features as patient age, sex, site of tumor, clinical presentation and tumor stage and the germinal center differentiation in DLBCL (germinal center B-cell (GCB) or activated B-cell (ABC)) and the double/triple genetic hits status.Material and Methods: The material of this study comprised 52 formalin-fixed paraffin- embedded blocks representative of diffuse large B cell lymphoma cases retrieved from the archival material available at the Pathology Department, Faculty of Medicine, Alexandria University. Subclassification of cases into (germinal center / and post-germinal center types) using a panel of immunohistochemical markers including CD10, BCL6 and MUM1 was done and scoring of double/triple hit expression using a panel of immunohistochemical markers including c-MYC, BCL6 and BCL2 was also done and correlation with their subtypes and clinical data was performed.Results: there was significant association between germinal center differentiation and tumor stage (MCp= 0.018).Conclusion: Germinal center differentiation status is a valid method to evaluate the prognosis of DLBCL.

Evaluation of Signal Transducer and Activator of Transcription 3 (STAT-3) Protein Expression in Non-Hodgkin Lymphoma Cases in Hospital USM.

Evolving targeted therapy on Janus Associated Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway, especially pertaining to STAT-3 protein in non-Hodgkin lymphoma (NHL), provides new treatment strategies. STAT-3 protein also relates to the prognostication of NHL. Hence, we aimed to evaluate the expression of STAT-3 protein in NHL cases diagnosed in Hospital Universiti Sains Malaysia (USM). A retrospective cross sectional study using formalin fixed paraffin embedded (FFPE) tissue blocks of 95 NHL cases were obtained. STAT-3 immunostaining was performed and evaluated. The proportion and association between the expression of STAT-3 protein with subtypes of NHL were statistically analyzed. The majority of the cases (78.9%) had positive STAT-3 protein expression. 64.2% were among aggressive B cell NHL, whilst 20.0% of them were diffuse large B cell lymphoma, a non-germinal center B subtype (DLBCL-NGCB). There is also an association between STAT-3 protein expression with DLBCL subtypes (p = 0.046). Our study demonstrated a remarkable expression of STAT-3 protein in NHL, in which DLBCL subtypes had significant association. A larger scale study with a combination of JAK protein evaluation should be undertaken in the future.

Characterization of extracellular vesicles in AIDS-associated non-Hodgkin lymphoma.

Abstract Even with the use of anti-retroviral therapy, HIV +subjects develop non-Hodgkin lymphoma (NHL) at a higher frequency than uninfected subjects. Extracellular vesicles (EVs) secreted by cells may contain immunomodulatory molecules; for example, tumor cells can secrete EVs containing bioactive PD-L1 that can inhibit anti-tumor responses. We have shown EVs bearing PD-L1, TNF-RII or IL-6Rα can be biomarkers of NHL risk. Here we aim to characterize EVs isolated from EBV +and EBV −lymphoma cell lines to define their involvement in lymphomagenesis and identify more EV-associated biomarkers for NHL risk (like immune checkpoint molecules CD80, CD86, CD28). EVs were isolated from AIDS-NHL and NHL cell lines using a total exosome isolation kit and analyzed by Western blot. A subset of these were also analyzed by proteomics using Metascape, Reactome, and R. EVs were also isolated from archived serum of HIV +individuals before NHL onset (n = 51) and matched HIV +controls (n = 53) from participants of the MACS/WIHS Combined Cohort study, with a total exosome isolation kit and characterized by Western blot. EV-specific markers were detected in EVs isolated from cell lines and serum. EVs isolated from EBV +cell lines largely expressed LMP1, an EBV CD40 homolog that induces EBV-driven B cell transformation. In EVs isolated from serum, levels of CD80, CD86, or CD28 were comparable between pre-NHL samples and matched controls, though control samples tended to have greater CD80 and CD86 expression. Our findings shed light on the EV marker expression profiles of AIDS-NHL and NHL cell lines and show LMP1 expression in EBV +cell lines. Additionally, this research provides initial insight into the complex composition of EVs and their role in lymphomagenesis. Supported by grants from NIH (R01CA228157, P30CA016042-S, R01CA228157S)

Patient Characteristics and Outcomes of Outpatient Tisagenlecleucel Recipients for B Cell Non-Hodgkin Lymphoma

Tisagenlecleucel (tisa-cel) is an approved CD19-directed chimeric antigen receptor T cell (CAR-T) therapy for relapsed/refractory B cell malignancies. Given potentially life-threatening toxicities, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, inpatient tisa-cel infusion and toxicity monitoring are often considered; however, the toxicity profile of tisa-cel may be conducive to outpatient administration. Here we review the characteristics and outcomes of tisa-cel recipients treated in the outpatient setting. Patients age ≥18 years with B cell non-Hodgkin lymphoma who received tisa-cel between June 25, 2018, and January 22, 2021, at 9 US academic medical centers were included in a retrospective analysis. Six of the 9 representative centers (75%) had an outpatient program in place. A total of 157 patients were evaluable, including 93 (57%) in the outpatient treatment group and 64 (43%) in the inpatient treatment group. Baseline characteristics, toxicity and efficacy, and resource utilization were summarized. The most common lymphodepletion (LD) regimen was bendamustine in the outpatient group (65%) and fludarabine/cyclophosphamide (91%) in the inpatient group. The outpatient group had more patients with a Charlson Comorbidity Index of 0 (51% versus 15%; P < .001), fewer patients with an elevated lactate dehydrogenase (LDH) level above the normal range at the time of LD (32% versus 57%, P=.003) compared to the inpatient group, and a lower Endothelial Activation and Stress Index score (.57 versus 1.4; P < .001). Any-grade CRS and ICANS were lower in the outpatient group (29% versus 56% [P < .001] and 10% versus 16% [P=.051], respectively). Forty-two outpatient tisa-cel recipients (45%) required an unplanned admission, with a median length of stay of 5 days (range, 1 to 27 days), compared to 13 days (range, 4 to 38 days) in the inpatient group. The median number of tocilizumab doses administered was similar in the 2 groups as were the rate of intensive care unit (ICU) transfer (5% versus 8%; P=.5) and median length of ICU stay (6 days versus 5 days; P=.7). There were no toxicity-related deaths in the 30 days post-CAR-T infusion in either group. Progression-free survival and overall survival were similar in the 2 groups. With careful patient selection, outpatient tisa-cel administration is feasible and associated with similar efficacy outcomes as inpatient treatment. Outpatient toxicity monitoring and management may help optimize healthcare resource utilization.

Treatment Outcome of Burkitt's Lymphoma in Adolescents and Adults: A Retrospective Study

Abstract Introduction Burkitt's lymphoma (BL) is a highly aggressive B cell non-Hodgkin lymphoma (NHL) having three distinct subtypes: endemic, sporadic, and immunodeficiency-associated BL. Sporadic BL accounts for only 1 to 2% of adult NHL. Objectives The objective of this article was to study the clinical profile and treatment outcome of patients with BL. Materials and Methods This was a retrospective study of 60 patients with BL conducted in the department of medical oncology at a tertiary cancer center in India during a 10-year period. Patients with BL/leukemia above 14 years of age diagnosed during the study period were included and their clinical presentation, treatment details, and outcome were studied. Results Among 60 cases with BL, there were 41 males and 19 females. The median age at presentation was 42 years (range: 14–81 years). The main symptoms were lymphadenopathy, abdominal pain, and abdominal distension. Two patients each had paraparesis, breast lump, and jaw swelling and one patient had involvement of the cervix. Thirteen patients had features of tumor lysis at presentation. The Ann Arbor stage was I in 17, II in 16, III in 5, and IV in 22. Fifty-five patients received combination chemotherapy that included hyper-cyclophosphamide, vincristine, adriamycin, dexamethasone ± rituximab (hyper-CVAD ± R; 35), cyclophosphamide, adriamycin, vincristine, prednisolone/ cyclophosphamide, vincristine, prednisolone ± rituximab CHOP ± R (13), Berlin-Frankfurt-Munich protocol (4), and others (3). Thirty-four patients attained remission, 13 patients had progressive disease, and 8 patients died during chemotherapy. At a median follow-up of 113 months, 58% patients were alive. Conclusions BL accounts for 1.57% of NHL above the age of 14 years with male preponderance. Intensive, short-duration chemotherapy is the standard treatment. Treatment with hyper-CVAD ± R gives 8-year progression-free survival and overall survival (OS) of 60%. Treatment with CHOP ± R is an alternative option in elderly frail patients with an 8-year OS of 46%.

Abstract CT130: An open label, dose escalation, phase 1 study of AT101, a novel CD19-directed CAR-T cell therapy targeting a membrane-proximal epitope of CD19, in patients with relapsed or refractory B cell non-Hodgkin lymphoma

Abstract Background and Preliminary Data: All the FDA-approved CD19 CAR-T cell therapies are based on an antigen-binding domain (scFv) based on the FMC63 antibody which binds to the membrane-distal region of CD19 to an epitope encoded by exons 3 and 4 (Klesmith JR, Biochemistry, 2019; Zhang Z, JITC, 2020). While these CART19 products are very effective in the clinic, the majority of patients still do not respond or eventually relapse due to several mechanisms of resistance, including T cell dysfunction and epitope CD19-negative escape. Novel strategies to enhance the activity of CART cells and reduce escape are critically needed. We recently demonstrated that modifications of the binding region of the CAR (scFv) (Singh N., Nat Med, 2021) can drastically change the interaction between the CAR T cell and the cancer cells, potentially improving the anti-tumor effect. To this goal, we developed a novel anti-CD19 antibody clone (1218) that binds to a membrane-proximal epitope of CD19 (exon 2 region K59-K63) thereby not competing with FMC63. We developed a novel CART19, called AT101, using a humanized 1218 scFv along with 4-1BB costimulatory and CD3zeta domain in a lentiviral backbone. In preclinical models, AT101 showed more potent in vitro cytotoxicity against CD19-positive B lymphoma cells in a long-term killing assay and in a B-ALL (NALM6) in vivo model as compared to the control of FMC63 based CAR-T cells. In addition, differently than FMC63-based CART, AT101 could target tumor cells expressing point mutations of CD19 that are associated with relapse post-CART19 (FMC63) (Zhang Z, JITC, 2020) and leukemic blasts aberrantly expressing FMC63 CAR19 on their surface (Ruella M, Nat Med, 2018). Based on the preclinical efficacy and safety, a phase 1 clinical trial testing autologous AT101 was started for patients with relapsed and refractory B-cell non-Hodgkin lymphoma. Trial Design and Methods: This open-label, multi-center, first-in-human Phase 1 study will assess the safety and feasibility of AT101 in patients with relapsed or refractory B cell non-Hodgkin lymphoma. Key eligibility criteria include patients aged ≥19 years of age with histologically confirmed relapsed or refractory aggressive B-cell non-Hodgkin lymphoma. In this phase 1 trial, patients (n=3 per dose level; up to n=18 in total) are treated with AT101 in 3 dose-escalation cohorts based on a standard 3 + 3 design. CART doses are 2.0 × 105, 1.0 × 106, or 5.0 × 106 CAR+T cells/kg. The primary objective is to determine the safety, the maximum tolerated dose (MTD), and the recommended phase 2 dose (RP2D) of AT101 in participants following lymphodepletion with cyclophosphamide and fludarabine (250 mg/m2 and 25 mg/m2). The secondary objective is to evaluate the preliminary efficacy assessments (overall response rate (ORR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), event-free survival (EFS), and pharmacokinetics of AT101. Exploratory objectives include assessment of CD19 expression and cytokines in the blood. Patients will be followed for safety for at least 60 months post AT101 infusion. Clinical trial registry number: NCT05338931. As of January 11, 2023, AT101 has been infused to six patients in cohort 1 and three patients in cohort 2. Detailed results will be presented at the meeting. Citation Format: Yunlin Zhang, Ki Hyun Kim, Dok Hyun Yoon, Ruchi P. Patel, Jae-Cheol Jo, Hyungwoo Cho, Jong-Ho Lee, Hyun-Jong Lee, Lei-Guang Cui, In-Sik Hwang, Young Ha Lee, Jong-Hoon Kim, Yong Gu Lee, Puneeth Guruprasad, Jong-Seo Lee, Junho Chung, Marco Ruella. An open label, dose escalation, phase 1 study of AT101, a novel CD19-directed CAR-T cell therapy targeting a membrane-proximal epitope of CD19, in patients with relapsed or refractory B cell non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT130.