Diffuse Large Cell Research Articles

Abstract 2531: FCN-683, a novel second-generation BCL-2 inhibitor, is highly potent, selective and efficacious against clinically relevant venetoclax-resistant mutations

Abstract B-cell lymphoma 2 (BCL-2) is a member of the anti-apoptotic BCL-2 family and is dysregulated in B-cell malignancies, including chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML) and Non-Hodgkin’s lymphoma. Venetoclax (VEN), also known as ABT-199, is the only BCL-2 inhibitor approved by FDA for treating CLL/SLL and AML. VEN is a BH3-mimetic drug competitively inhibits BCL-2 by binding to the hydrophobic groove and thereby displacing proapoptotic proteins and inducing apoptosis. Despite high effectiveness of the initial treatment, most patients treated with VEN eventually relapse or develop resistance. An important resistance mechanism is acquisition of BCL-2 G101V mutation, detected in about 50% of CLL patients 19-42 months after receiving VEN. Other resistance mutations identified clinically so far include D103, A113G, R129L, and V156D, etc. Overcoming VEN-resistant Bcl-2 mutations represent an unmet medical need. Here, we introduce a novel and selective second-generation oral BCL-2 inhibitor, FCN-683, with sub-nano or single nanomolar affinity to BCL-2 and VEN-resistant BCL-2 mutations including G101V, D103E/V/Y, F104L, A113G, R129L and V156D. FCN-683 showed much higher affinity to BCL-2 than BCL-xL, suggesting its good potential to avoid BCL-xL-linked thrombocytopenia. FCN-683 exhibited comparable or superior anti-proliferation activities compared with VEN in various BCL-2-addicted human B-cell lymphoma cells, including DOHH2 follicular lymphoma cells, Toledo diffuse large B-cell lymphoma cells, and RS4;11 acute lymphoblastic leukemia cells, while sparing BCL-xL-dependent cells. Of note, FCN-683 displayed potent anti-proliferative activities against a panel of genetically engineered RS4;11 cells ectopically expressing VEN-resistant BCL-2 mutants such as G101V, D103 or several other mutants above-mentioned, outperforming VEN. Correspondingly, FCN-683 significantly inhibited tumor growth in mice xenografts derived from human B-cell lymphomas harboring BCL-2 or various VEN-resistant BCL-2 mutations and its anti-tumor efficacy was much superior to VEN. In non-clinical studies, FCN-683 possessed excellent pharmacokinetic (PK) properties comparable to VEN. Preferable safety profiles of FCN-683 were shown with no potential hERG inhibitory effect and less drug-drug interaction potential, as evidenced by no inhibitory effect (IC50 >50 μM) on CYP2C9 enzyme compared with VEN (IC50 1.05 μM). Together, FCN-683 is highly potent, selective and highly efficacious against a variety of clinically relevant VEN-resistance BCL-2 mutations in vitro and in vivo and exhibits favorable PK and safety profiles, highlighting its therapeutic potential to become an effective therapeutic approach for VEN-naïve or -resistant BCL-2-addicted B-cell malignancies. Citation Format: Shu (Sophie) Lin, Hongbin Liu, Rui Tan, Weipeng Zhang, Chengxi He, Yue Rong, Zhuo Huang, Jinhua Yu, Yunling Wang, Yangli Qi, Xingdong Zhao, Lihua Jiang, Yanxin Liu, Xilei Wang, Zongyao Zou, Jing Sun, Yuwei Gao, Weibo Wang, Xiaohui Guan, Yifang Wu. FCN-683, a novel second-generation BCL-2 inhibitor, is highly potent, selective and efficacious against clinically relevant venetoclax-resistant mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2531.

Ras interacting protein 1 facilitated proliferation and invasion of diffuse large B-cell lymphoma cells

ABSTRACT A GTPase binding protein, Ras interacting protein 1 (RASIP1), has been reported with a tumor-promoting role in lung cancer cells, and its role in lymphoma remains unknown. The analysis of medical databank shows that RASIP1 is upregulated in diffuse large B-cell lymphoma (DLBCL) specimens. In this article, we demonstrated that RASIP1 is highly expressed in DLBCL cell lines, compared with primary B cells. The gain- and loss-of-function experiments were performed to investigate the effects of RASIP1 on DLBCL cells. CCK-8, flow cytometry, western blot, and transwell assays demonstrated that silence of RASIP1 inhibited proliferation, cell cycle transition, and invasion and induced significant apoptosis in DLBCL cells, and ectopic expression of RASIP1 played opposite roles. Xenograft results revealed that RASIP1 facilitated the growth of DLBCL cells in vivo. These findings suggest that RASIP1 may be required for malignancy of DLBCL cells. In addition, we also found that the expression of RASIP1 was negatively regulated by forkhead box O3 (FOXO3), which has been reported to suppress the proliferation of DLBCL cells. Our results indicate that FOXO3 is bound to the promoter sequence of RASIP1 and inhibits its transcription. The suppressive effects of FOXO3 on proliferation and invasion of DLBCL cells were neutralized by RASIP1. In conclusion, we demonstrate that FOXO3 negatively regulated RASIP1 facilitates growth and invasion of DLBCL cells, provides novel diagnostic markers and therapeutic targets for DLBCL in clinic.

Transcriptome Analysis of Diffuse Large B-Cell Lymphoma Cells Inducibly Expressing MyD88 L265P Mutation Identifies Upregulated CD44, LGALS3, NFKBIZ, and BATF as Downstream Targets of Oncogenic NF-κB Signaling.

During innate immune responses, myeloid differentiation primary response 88 (MyD88) functions as a critical signaling adaptor protein integrating stimuli from toll-like receptors (TLR) and the interleukin-1 receptor (IL-1R) family and translates them into specific cellular outcomes. In B cells, somatic mutations in MyD88 trigger oncogenic NF-κB signaling independent of receptor stimulation, which leads to the development of B-cell malignancies. However, the exact molecular mechanisms and downstream signaling targets remain unresolved. We established an inducible system to introduce MyD88 to lymphoma cell lines and performed transcriptomic analysis (RNA-seq) to identify genes differentially expressed by MyD88 bearing the L265P oncogenic mutation. We show that MyD88L265P activates NF-κB signaling and upregulates genes that might contribute to lymphomagenesis, including CD44, LGALS3 (coding Galectin-3), NFKBIZ (coding IkBƺ), and BATF. Moreover, we demonstrate that CD44 can serve as a marker of the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) and that CD44 expression is correlated with overall survival in DLBCL patients. Our results shed new light on the downstream outcomes of MyD88L265P oncogenic signaling that might be involved in cellular transformation and provide novel therapeutical targets.

PROTAC Linkerology Leads to an Optimized Bivalent Chemical Degrader of Polycomb Repressive Complex 2 (PRC2) Components.

Bivalent chemical degraders, otherwise known as proteolysis-targeting chimeras (PROTACs), have proven to be an efficient strategy for targeting overexpressed or mutated proteins in cancer. PROTACs provide an alternative approach to small-molecule inhibitors, which are restricted by occupancy-driven pharmacology, often resulting in acquired inhibitor resistance via compensatory increases in protein expression. Despite the advantages of bivalent chemical degraders, they often have suboptimal physicochemical properties and optimization for efficient degradation remains highly unpredictable. Herein, we report the development of a potent EED-targeted PRC2 degrader, UNC7700. UNC7700 contains a unique cis-cyclobutane linker and potently degrades PRC2 components EED (DC50 = 111 nM; Dmax = 84%), EZH2WT/EZH2Y641N (DC50 = 275 nM; Dmax = 86%), and to a lesser extent SUZ12 (Dmax = 44%) after 24 h in a diffuse large B-cell lymphoma DB cell line. Characterization of UNC7700 and related compounds for ternary complex formation and cellular permeability to provide a rationale for the observed improvement in degradation efficiency remained challenging. Importantly, UNC7700 dramatically reduces H3K27me3 levels and is anti-proliferative in DB cells (EC50 = 0.79 ± 0.53 μM).

Orthovoltage X-Ray Therapy of Large Cell Invasive Nasal Lymphoma in Cat

Abstract Feline nasal lymphoma is generally a localized radiosensitive tumor. Treatment options include radiation therapy, chemotherapy, or a combination of both modalities. Radiation therapy (RT) in a combination with chemotherapy lead to median survival from 19 months to 955 days. The objective of this paper is to report a case of a large cell late stage invasive feline nasal lymphoma and to evaluate the results of orthovoltage radiation therapy and its side effects. A 12-year-old female mixed breed cat was presented with nasal discharge, sneezing and hyporexia. Definitive histopathology diagnosis was diffuse large cell lymphoma. Computed tomography revealed an advanced stage of the disease, an intranasal mass, bone lysis, invasion of the orbital space and central nervous system. A radical course of X-ray therapy was performed (SFD = 4 Gy, TSD = 40 Gy, 10 sessions, 3 times a week). Radiation side effects were well tolerated and resolved with supportive treatment. Lymphoma is a systemic process and requires a multidisciplinary approach. On most cases, the penetrating ability of radiation therapy of 3.5 cm in depth is enough for small domestic animals. A significant effect is noted in the middle of the treatment course. The fractionated regime did not cause early radiation damage. The relapse-free period was up to 8 months. The patient had a satisfying quality of life for 5 months. Orthovoltage X-ray therapy can be considered as a treatment choice for local nasal lymphoma. X-ray therapy is a widely used modality in veterinary medicine considering the price of machines and costs of treatment.

HIF-1α mediates CXCR4 transcription to activate the AKT/mTOR signaling pathway and augment the viability and migration of activated B cell-like diffuse large B-cell lymphoma cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy with a high relapse rate. We previously found that C-X-C motif chemokine receptor 4 (CXCR4) was highly expressed in DLBCL and associated with poor prognosis. This study focused on the effect of hypoxia-inducible factor-1α (HIF-1α) on CXCR4 expression and the DLBCL progression. Two activated B cell-like DLBCL cell lines Ly-3 and SUDHL2 were transfected with overexpression and knockdown plasmids or HIF-1α. The viability and migration of DLBCL cells were significantly increased under hypoxic conditions, or upon HIF-1α overexpression under normoxic conditions, but the HIF-1α downregulation led to inverse trends. However, the promoting effects of HIF-1α overexpression on DLBCL cells were suppressed by Plerixafor (a CXCR4 inhibitor). The luciferase and chromatin immunoprecipitation assays revealed that HIF-1α bound to the functional site HRE1 on CXCR4 promoter to activate its transcription. HIF-1α-mediated CXCR4 activation further led to increased phosphorylation of AKT/mTOR under hypoxic conditions. Taken together, this work reports that HIF-1α promotes viability and migration of activated B cell-like cells under hypoxia, which might involve the transcription of CXCR4 and the activation of the AKT/mTOR pathway. The finding may provide novel lights in the management of DCBCL.

Management of orbital diffuse large cell b lymphoma: a case report

Orbital lymphoma is a very rare location of large cell lymphomas. It accounts for 10% of orbital tumors. Proptosis is the most frequent sign, but it should be noted that the clinical presentation depends greatly on the initial site of the tumor. The diagnosis is suspected on clinical and radiological elements and confirmed by anatomopathologica exam. Chemotherapy is the main treatment in the management of orbital lymphoma, often combined with radiation therapy and surgery. We present a case of orbital diffuse large cell B lymphoma treated with chemothérapy with RCHOP protocol and external radiothérapy, and we discusse current recommendations from the literature

Oncogenic mutations in the DNA-binding domain of FOXO1 that disrupt folding: Quantitative insights from experiments and molecular simulation.

FOXO1, a member of the family of winged-helix motif Forkhead box (FOX) transcription factors, is the most abundantly expressed FOXO member in mature B cells. Sequencing of diffuse large B-cell lymphoma (DLBCL) tumors and cell lines identified specific mutations in the forkhead domain linked to loss of function. Differential scanning calorimetry and thermal shift assays were used to characterize how eight of these mutations affect the stability of the FOX domain. Mutations L183P and L183R were found to be particularly destabilizing. Electrophoresis mobility shift assays show these same mutations also disrupt FOXO1 binding to their canonical DNA sequences, suggesting that the loss of function is due to destabilization of the folded structure. Computational modeling of the effect of mutations on FOXO1 folding was performed using alchemical free energy perturbation (FEP), and a Markov model of the entire folding reaction was constructed from massively parallel molecular simulations, which predicts folding pathways involving the late folding of helix α3. Although FEP can qualitatively predict the destabilization from L183 mutations, we find that a simple hydrophobic transfer model, combined with estimates of unfolded-state solvent-accessible surface areas from molecular simulations, is able to more accurately predict changes in folding free energies due to mutations. These results suggest that the atomic detail provided by simulations is important for the accurate prediction of mutational effects on folding stability. Corresponding disease-associated mutations in other FOX family members support further experimental and computational studies of the folding mechanism of FOX domains.

Effect of MiR-424-5p on the Drug Resistance of Diffuse Large B-Cell Lymphoma Cells by Regulating PD-1/PD-L1 Signaling Pathway

To explore the effect of microRNA-424-5p (miR-424-5p) on the drug resistance of diffuse large B-cell lymphoma (DLBCL) cells by regulating the programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway. Human DLBCL cell line CRL2631 cells were induced to construct CRL2631-CHOP resistant cell line. RT-qPCR and Western blot were used to detect the expression levels of MiR-424-5p, PD-L1 mRNA and protein, and multidrug resistance gene-1 (MDR-1) protein in CRL2631 cells and CRL2631-CHOP cells, respectively. The target genes of MiR-424-5p was verified by dual luciferase reporter assay. The miRNA simulation/interference technology and thiazole blue (MTT) method were used to detect the resistance of CRL2631 cells and CRL2631-CHOP cells to CHOP. Compared with CRL2631 cells, the drug resistance of CRL2631-CHOP cells to CHOP and the levels of MDR-1 protein (P<0.05), PD-L1 mRNA and protein in the cells were significantly increased (both P<0.001), while the relative level of MiR-424-5p was significantly reduced (P<0.001). The result of the dual luciferase reporter assay showed that PD-L1 was the direct downstream target gene of MiR-424-5p (P<0.001). After transfection of MiR-424-5p inhibitor, the resistance of CRL2631 cells to CHOP drugs increased, and the expression level of MDR-1 protein (P<0.01), PD-L1 mRNA and protein also increased significantly (both P<0.01). After transfection of MiR-424-5p mimics, the resistance of CRL2631-CHOP cells to CHOP drugs decreased, and the expression level of MDR-1 protein (P<0.001), PD-L1 mRNA and protein also decreased significantly (both P<0.001). Overexpression of PD-L1 could reverse the inhibitory effect of upregulating MiR-424-5p on PD-L1 (P<0.001). Down-regulation of MiR-424-5p enhances the drug resistance of DLBCL cells by regulating the PD-1/PD-L1 signaling pathway.

Baseline volumetric parameters of PET/CT &lt;sup&gt;18&lt;/sup&gt;F-FDG and international prognostic index at patients with diffuse B-cell large cell lymphoma

INTRODUCTION: Metabolic tumor volume (MTV) and Tumor lesion glycolysis (TLG) are used in 18F-fluorodeoxyglucose Positron-Emission Tomography combined with Computed Tomography (18F-FDG PET/CT) as biometric markers in whole tumor. International prognostic index (IPI) has been used during 30 years at patients with diffuse B-cell large cell lymphoma (DLBCL) for prognosis, use MTV and TLG was unknown. Our aims were to determinate the best statistical relationship between MTV, TLG and IPI in patients with DLBCL.OBJECTIVE: Complex study of baseline volumetric parameters PET/CT with 18F-FDG at patients with DLBCL and assessment of their correlation with prognostic groups.MATERIALS AND METHODS: Baseline 18F-FDG PET/CT performed in 31 primary patients with DLBCL and evaluated SUVmax, MTV, TMTV, TLG, TTLG at different involved anatomic zones; correlation between PET biomarkers and prognostic groups of patients.Statistics: The research materials were subjected to statistical processing using the methods of correlation and regression analyses.RESULTS: SUVmax mean level didn’t discerned at patients of low and high-risk groups, but TMTV has been less in favorable prognostic, than in unfavorable groups (152±3.0 sm3 vs 890±178 sm3 p&lt;0,01); the same TTLG (1291±474 g/ml×sm3 vs 7640±2067 g/ml×sm3 , p&lt;0.01) and IMA (6.7±0.7 vs 9.2±0.6; p&lt;0,01). All volumetric parameters have been less at patients with supradiaphragmatic lymph nodes, than infradiaphragmatic lymph nodes and bones too. Significant positive correlation was found between the TMTV u TTLG (p=0.965; p&lt;0,001), between TMTV, TTLG and progressive disease risk (p=0.735; p=0.747; p&lt;0,001). The regression analysis was derived to estimate the connection PET biomarkers and progressive disease risk.DISCUSSION: The 30-year experience of using IPI in assessing the prognosis of patients with DLBCL has shown its insufficient effectiveness in many clinical situations, and the R-IPI prognostic model has been tested on a smaller number of patients and can only be considered additionally. We studied in detail the initial volumetric parameters of patients with DLBCL in different regions of the lesion and their relationship with the level of prognosis according to the IPI criteria. Our data on large values of volumetric parameters in the infradiaphragmatic areas and bones may be related to the worse prognosis in patients with these localizations. Further broader studies of the initial volumetric biomarkers of PET in DLBCL patients will contribute to the development of approximate boundaries of specific volumetric parameters for different tumor localizations.CONCLUSION: The results obtained showed that the baseline volumetric parameters PET/CT at patients with DLBCL have significant connection with prognostic groups of patients and can be additional factors to influence of tactics of therapy.

Expression and Significance of Epstein-Barr Virus-Latent Membrane Protein 1 in Acquired Immune Deficiency Syndrome-Related Diffuse Large B-Cell Lymphoma

We aimed to investigate the expression and significance of Epstein-Barr virus-latent membrane protein 1 in human immunodeficiency virus-related diffuse large B-cell lymphomas. 22 cases of human immunodeficiency virus-related diffuse large B-cell lymphomas samples were collected. The control group consisted of 7 cases of non-human immunodeficiency virus-related diffuse large B-cell lymphomas, 6 cases of human immunodeficiency virus-reactive hyperplasia and 6 cases of non-human immunodeficiency virus. The expression of Epstein-Barr virus-latent membrane protein 1 was detected by immunohistochemistry and analyzed in combination with clinicopathology characteristics. The total positive rate of Epstein-Barr virus-latent membrane protein 1 was 77.3 % in human immunodeficiency virus-related diffuse large B-cell lymphomas and 71.4 % in non-human immunodeficiency virus-related diffuse large B-cell lymphomas. Most related diffuse large B-cell lymphomas cells showed nuclear staining pattern and the nuclear positive rate was 82.4 % in human immunodeficiency virus-related diffuse large B-cell lymphomas and 60 % in non-human immunodeficiency virus-related diffuse large B-cell lymphomas. There was no significant difference between human immunodeficiency virus-related diffuse large B-cell lymphomas and non-human immunodeficiency virus-related diffuse large B-cell lymphomas. The expression of Epstein-Barr virus-latent membrane protein 1 was significant between human immunodeficiency virus-related diffuse large B-cell lymphomas and benign lesion. Epstein-Barr virus-latent membrane protein 1 expression in non-germinal center B cell human immunodeficiency virus-related diffuse large B-cell lymphomas was significantly higher than that in germinal center B cell human immunodeficiency virus-related diffuse large B-cell lymphomas. However, Epstein-Barr virus-latent membrane protein 1 expression had no correlation with sex, age, location of tumor and clinical stage (p>0.05). Epstein-Barr virus-latent membrane protein 1 was mainly located in nuclear and overexpressed in human immunodeficiency virus-related diffuse large B-cell lymphomas and non-human immunodeficiency virus-related diffuse large B-cell lymphomas, suggesting that Epstein-Barr virus-latent membrane protein 1 overexpression was involved in tumourogenesis of related diffuse large B-cell lymphomas. Epstein-Barr virus-latent membrane protein 1 expression was correlated with immunophenotype of related diffuse large B-cell lymphomas. As the prognosis was different in different immunophenotype subgroups, Epstein-Barr virus-latent membrane protein 1 may be the potential marker of prognosis for human immunodeficiency virus-related diffuse large B-cell lymphomas.

In Vitro Diffuse Large B-Cell Lymphoma Cell Line Models as Tools to Investigate Novel Immunotherapeutic Strategies

Despite the high incidence of diffuse large B-cell lymphoma (DLBCL), its management constitutes an ongoing challenge. The most common DLBCL variants include activated B-cell (ABC) and germinal center B-cell-like (GCB) subtypes including DLBCL with MYC and BCL2/BCL6 rearrangements which vary among each other with sensitivity to standard rituximab (RTX)-based chemoimmunotherapy regimens and lead to distinct clinical outcomes. However, as first line therapies lead to resistance/relapse (r/r) in about half of treated patients, there is an unmet clinical need to identify novel therapeutic strategies tailored for these patients. In particular, immunotherapy constitutes an attractive option largely explored in preclinical and clinical studies. Patient-derived cell lines that model primary tumor are indispensable tools that facilitate preclinical research. The current review provides an overview of available DLBCL cell line models and their utility in designing novel immunotherapeutic strategies.

Primary cutaneous blastic marginal zone lymphoma: A comprehensive clinical, light microscopic, phenotypic and cytogenetic appraisal

BackgroundPrimary cutaneous marginal zone lymphoma (PCMZL) is a form of indolent lymphoproliferative disease where the disease is largely a cutaneous confined process. It is typically a neoplasm composed of post germinal small B-cells and light chain restricted plasma cells in a background of reactive T-cell hyperplasia and benign germinal centers. Rarely a significant degree of large cell infiltration occurs warranting the categorization as blastic marginal zone lymphoma. Materials and methodsWe reviewed our data base over a time period of 2016 to 2022 for cases diagnosed as blastic MZL. Twelve cases were identified. The clinical records and pathological data were reviewed. ResultsNine of the cases represented de novo forms of blastic MZL while in three cases there was a prior history of MZL. Multifocal cutaneous disease was not uncommon and one quarter of the cases had evidence of extracutaneous dissemination. All patients except three achieved remission with varied therapeutic interventions depending on the extent of the disease ranging from conservative re-excision to chemotherapy. No patient died from lymphoma. Light microscopically, there was evidence of a background of conventional MZL in the majority of cases. The large cell component was typically characterized by multiple micronodular aggregates throughout the dermis although in three cases there was a striking diffuse large cell component as the dominant infiltrate. Phenotypically, a third of the cases showed either CD5 or CD23 positivity amidst neoplastic B cells. Significant staining for BCL-2 was noted in the majority of cases tested while extensive MUM-1 positivity was observed in half of the cases tested. Kappa or lambda light chain restriction was seen in most. The Ki67 proliferation index exceeded 30 % in all cases. There was C-MYC positivity in two cases. While most cases did not detect cytogenetic abnormalities, one case had multiple cytogenetic hits that are associated with diffuse large B cell lymphoma. Next generation sequencing showed a Ten-eleven translocation 2 mutation in the earlier biopsy prior to transformation and in the later biopsy after transformation along with an additional B2M mutation in the transformed biopsy. Both types of mutations are very uncommon but held to contribute to tumor progression in the setting of diffuse large B cell lymphoma. ConclusionBlastic MZL is associated with a more aggressive clinical course. Even when there is disseminated disease patients while not always cured did not have a fatal course in this series. The light microscopic findings are reproducible. The background of MZL, identification of larger cells in significant numbers without a follicle center phenotype, at times expressing CD5 or CD23 with variable positivity for MUM1, BCL-2 and C-MYC and a high proliferation index define the pathology in most. Certain cytogenetic abnormalities and genetic mutations implicated in large cell transformation into a diffuse large B cell lymphoma are seen in blastic MZL with earlier biopsies prior to transformation potentially harboring at risk genetic mutations.

Effect of miR-27b on the proliferation and apoptosis of diffuse large b-cell lymphoma cells by targeting the regulation of MET/PI3K/AKT pathway

BackgroundThis study aimed to explore the regulation of miR-27b expression on MET/PI3K/AKT pathway, and to explain its effect on biological functions of DLBCL cells.MethodsThe expressions of miR-27b and MET gene in DLBCL cells and normal human B cell lines were determined by qRT-PCR. miR-27b expression in DLBCL cell line Toledo was over-expressed with the cell transfection method. The proliferation of DLBCL cells was determined by MTT. And the invasiveness of DLBCL cells was determined by Transwell. The level of apoptosis in DLBCL cells was determined by ELISA. miR-27b targeting of MET was verified by dual- luciferase reporter assay. The activation of MET/PI3K/AKT pathway and the expression of downstream related proteins were determined by Western blot.ResultsThe results showed that miR-27b was poorly expressed in DLBCL cell lines compared with normal human B cell lines, and was associated with its high proliferation, high invasiveness and low apoptosis level. High miR-27b expression can reduce the proliferation and increase the apoptosis level in DLBCL cells. By examining the effect of miR-27b over-expression on the MET/PI3K/AKT pathway, it was found that miR-27b can inhibit the proliferation and invasiveness and promote the apoptosis of DLBCL cells by targeting the inhibition of MET expression and the activation of PI3K/AKT pathway.ConclusionmiR-27b can inhibit the proliferation and invasiveness of DLBCL cells and promote the apoptosis of the cells by targeting MET/PI3K/AKT pathway.

Novel reconstruction method by mega-prosthesis wrapped with vancomycin-containing cement after resection of malignancies.

To introduce wrapping vancomycin-containing cement around a mega-prosthesis (MP) as a novel method to prevent prosthetic joint infection after reconstruction surgery for malignant bone and soft tissue tumors. Five patients with malignant bone and soft tissue tumors treated at our hospital from April 2009 to December 2019 were included. The average age was 71.4 years. Four males and one female were included. Three patients had a bone tumor, and two had a soft tissue tumor. Three right thighs and two left femurs were affected. These tumors were identified histologically as undifferentiated pleomorphic sarcoma, spindle cell sarcoma, diffuse large cell B-cell lymphoma, metastasis of renal cancer, and metastasis of lung cancer. All patients underwent tumor resection and reconstruction with a MP. In all cases, vancomycin-containing cement (2 g/40 g) was wrapped around the implant at the extension. The average follow-up period was 30.4 months. We surveyed whether infection occurred after surgical treatment. We also investigated the Musculoskeletal Tumor Society score and clinical outcome. We observed no postoperative infection. One case of local recurrence was observed, and a hip dissection was performed. The Musculoskeletal Tumor Society score was 79.26 ± 1.26 (mean ± standard deviation) (range: 76-80.3). Three patients remained disease-free, one survived but with disease, and one died of disease. Wrapping vancomycin-containing cement around the MP may be a useful method of preventing postoperative joint infections.

Patterns of Lymphoproliferative Disorders in Ethiopia, Based on Single-Center Experience

Background: Similar to other resource-poor countries, the patterns of lymphoproliferative disorders (LPD) in Ethiopia and in our setup are not clearly known. Here, we describe our experience based on conventional histopathology diagnosis. Method: This is a retrospective data-based cross-sectional study from August 2018 to December 2021 at St. Paul's Millennium Medical College, Addis Ababa, Ethiopia. Histopathology reports of cases with diagnoses of LPDs were retrieved from the department of pathology electronic report archive. The diagnostic sample included excisional lymph node and or core tissue biopsy. The diagnosis was based on conventional histopathology (hematoxylin and eosin staining) of the sample. Pertinent data were extracted using a data collection sheet. It was reviewed and analyzed using SPSS-24. Result:A total of 168 patients with necessary documented information were identified; 109(64.9%) were males; the mean (range) age was 46 (16-86) years. The majority of the patients, 139 (82.7%) had Non-Hodgkin lymphomas (NHL) with major histological sub-types being Small lymphocytic lymphoma (24.5%) and Diffuse large cell lymphoma (15.8%). Hodgkin Lymphoma (HL) accounted for 17(10.1%) cases. Sub-classification as a Classical HL was possible in 13 (76.5%) cases(fig.1). But it was not able to sub-classify NHLs histologically in 48(34.5%) and HLs in 3(17.6%) of the cases. Specific lymphoma diagnosis was not possible in 12(7.1%) patients with lymphoproliferative disorders on which an atypical LPD diagnosis was rendered. The most common biopsied lymph node area was the cervical lymph node (33.3%) (table 1) while the most common extranodal site was gastrointestinal (11.9%). Conclusion: Our study indicates that accurate diagnosis and classification of lymphoproliferative disorders is limited in our setup. Therefore, Capacity building in ancillary tests as limited panel immunohistochemistry and quality improvement projects may help to alleviate the shortcomings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Value of Lymphopenia and Total Metabolic Tumor Volume in Diffuse Large Cell Lymphoma of B Phenotype

BACKGROUND: Diffuse large cell B lymphoma (DLBCL) is the most common lymphoma in adults. The International Prognostic Index (IPI) has been used since 1993 to assess the severity of the disease. Other prognostic factors have emerged such as total metabolic tumor volume (TMTV) assessed by PET-CT and lymphopenia at diagnosis. The objective of our study was to evaluate the prognostic value of lymphopenia and TMTV in LDGCB in comparison with the IPI score. METHOD: We constituted a retrospective monocentric cohort of patients treated with R-CHOP or R-ACVBP in 1st line of DLBCL. The TMTV on the initial PET-CT scan was acquired using an artificial intelligence module and was related to the patient volume measured on the scan (TMTV/PV ratio). The effect of prognostic factors on 3-year progression-free survival (PFS) and overall survival (OS) was analyzed by comparison of Kaplan-Meier curves (Log-Rank test) and Cox regression. RESULTS: 118 patients with a lymphocyte count at diagnosis were included in the survival analysis. Among them, 53 patients underwent PET-CT analysis. Two factors were associated with decreased OS at 3 years: lymphocyte count at diagnosis ≤ 0.7G/L (76.9% vs 93.5%; p = 0.009) and TMTV/VP ratio > 0.35% (81% vs 96.9%; p = 0.05). The combination of these two factors was superior to the IPI score in discriminating patients' 3-year PFS (88.9% if no adverse factors, 55.6% if 1 factor, 12.5% if 2 factors present; p < 0.001). CONCLUSION: Lymphopenia and elevated TMTV/VP ratio at the diagnosis of LDGCB are individually associated with decreased overall survival of patients. The combination of these two factors is superior to the IPI in our study to discriminate the 3-year progression-free survival of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase1/1b Dose Escalation/Dose Expansion Study of Prgn-3007 Ultracar-T Cells in Patients with Advanced Hematologic and Solid Tumor Malignancies

Background: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a promising target for cancer treatment as its expression enhances tumor cell growth and survival, promotes epithelial-mesenchymal transition and metastasis and participates in pro-survival pathways such as PI3-Kinase/AKT, EGFR signaling, and the p38-MAPkinase pathway. ROR1 is undetectable in normal adult tissues, however aberrant expression of ROR1 in malignancies has been detected in B-cell malignancies such as B-cell acute lymphoblastic leukemia (B-ALL), diffuse large cell B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL). Furthermore, upregulated expression has been detected in various solid tumors, including ovarian cancer, breast adenocarcinomas encompassing triple negative breast cancer (TNBC), pancreatic cancer, Ewing's sarcoma and lung adenocarcinoma. The increased expression of ROR1 in hematological and solid tumor malignancies has been associated with tumor proliferation, metastasis and poor clinical outcomes. The autologous UltraCAR-T® platform is designed to overcome the limitations of traditional CAR T which leads to an exhausted T cell phenotype, high manufacturing costs, and treatment delays, using advanced non-viral gene delivery system and a decentralized, overnight manufacturing process. Scientific Rationale: PRGN-3007 is built on Precigen's UltraCAR-T platform and is engineered to simultaneously express a ROR1-specific chimeric antigen receptor (ROR1 CAR); membrane bound interleukin-15 (mbIL15) for enhanced in vivo expansion and persistence; a kill switch for improved safety profile; and a novel mechanism for the intrinsic blockade of programmed cell death protein 1 (PD-1) gene expression. Preclinical studies with PRGN-3007 UltraCAR-T cells showed significant reduction in PD-1 expression with increased ROR1 specific cytotoxicity and inflammatory cytokine production upon co-culture with ROR1+ PD-L1+ hematological and solid tumor cells. Furthermore, PRGN-3007 was selectively and effectively eliminated by the kill switch activator antibody treatment. In vivo, a single administration of PRGN-3007, effectively reduced tumor burden and significantly improved overall survival (p<0.05) of tumor bearing mice compared to Control ROR1 CAR-T in an aggressive xenograft model of mantle cell lymphoma. Blood analyses demonstrated sustained downregulation of PD-1 expression, rapid expansion, long-term persistence, and a predominant central memory phenotype of PRGN-3007 in tumor bearing mice. (Blood (2021) 138 (Supplement 1): 1694.) Study Design: This is a first-in-human, Phase 1/1b dose escalation/dose expansion study to evaluate the safety and effectiveness of PRGN-3007 UltraCAR-T cells in patients with advanced ROR1-postive (ROR1+) hematologic (CLL, MCL, ALL, and DLBCL; Arm 1) and solid tumors (TNBC; Arm 2). Key inclusion criteria include absolute lymphocyte count ≥ 0.2k/µL, KPS > 70, adequate organ function, confirmed expression of ROR1 and: relapsed or refractory CLL; pathologically confirmed ALL B-cell precursor and measurable disease, pathologically confirmed MCL with overexpression of cyclin D1 or presence of t (11;14); pathologically confirmed DLBCL or high grade B-cell lymphoma including transformation from low grade lymphoma; or locally advanced unresectable or metastatic histologically confirmed TNBC with measurable disease as per RECIST 1.1. Study subjects undergo leukapheresis followed by lymphodepletion with either fludarabine 30mg/m2 and cyclophosphamide 500mg/m2 for 3 days (Arm 1) or 60 mg/kg cyclophosphamide for 2 days (Arm 2). The study will enroll in 2 phases: an initial dose escalation phase followed by a dose expansion phase. Up to 4 dose levels are planned in dose escalation phase (Table 1), and the MTD will be determined separately in subjects in the hematologic arm and the solid tumor arm. The MTD for each arm will be expanded independently in the dose expansion phase of the study. All subjects will be followed for adverse events, CAR-T-related toxicities, disease response and PRGN-3007 cell expansion and persistence. In addition, the mechanisms of safety and effectiveness of PRGN-3007 cells will be evaluated with correlative assays of specific immune response pathways. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Orally bioavailable BTK PROTAC active against wild-type and C481 mutant BTKs in human lymphoma CDX mouse models

AbstractBruton tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade. BCR activation can contribute to the growth and survival of B-cell lymphoma or leukemia. The inhibition of the BCR signaling pathway is critical for blocking downstream events and treating B-cell lymphomas. Herein, we report potent and orally available proteolysis-targeting chimeras (PROTACs) that target BTK to inactivate BCR signaling. Of the PROTACs tested, UBX-382 showed superior degradation activity for wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of half-maximal degradation concentration in diffuse large B-cell lymphoma cell line. UBX-382 was effective on 7 out of 8 known BTK mutants in in vitro experiments and was highly effective in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK–expressing TMD-8 cells over ibrutinib, ARQ-531, and MT-802. Remarkably, oral dosing of UBX-382 for <2 weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. UBX-382 also provoked the cell type–dependent and selective degradation of cereblon neosubstrates in various hematological cancer cells. These results suggest that UBX-382 treatment is a promising therapeutic strategy for B-cell–related blood cancers with improved efficacy and diverse applicability.

Real-world use and acceptance of biosimilar rituximab in oncology practice in India

Background: To describe the treatment patterns, patient characteristics and usage pattern of biosimilar rituximab for the treatment of Non-Hodgkin lymphomas (NHL) in India.Methods: This real-world, retrospective, analysis included adult patients receiving biosimilar rituximab between April 2021 and March 2022.Results: A total of 750 patients with NHL who received biosimilar rituximab were included. The most common indications reported in this analysis were diffuse large cell B-cell lymphoma [DLBCL, 64.5% (n=484)], follicular lymphoma, [FL, 23.7% (n=178)], and mantle cell lymphoma [MCL, 5.3% (n=40)]; other subtypes constituted 6.4% of the patients (n=48). The mean age of patients among DLBCL, FL and MCL was 65.5, 55.3 and 57 years, respectively. Across the lymphomas, &gt;90% of the patients received R-chemo as first-line therapy. R-CHOP was the most common regimen across indications.Conclusions: Biosimilar rituximab-based chemotherapy is being adopted in real‑world clinical practice in India for the management of patients with B-cell NHL including DLBCL, FL, MCL and other lymphomas.