Diffuse Large Cell Research Articles

Primary splenic diffuse large B-cell lymphoma (PS-DLBCL) presenting in an asymptomatic patient after sports trauma

Primary splenic diffuse large cell lymphoma is unusual and accounts for 2% of all splenic lymphomas. The symptoms included left upper abdominal pain, indigestion, loss of appetite, generalized weakness, fever, anemia, and thrombocytopenia or elevated C Reactive Protein. The current case report, to our knowledge the first of its kind, presents unusual clinicopathologic findings, where the patient remained asymptomatic until the time of diagnosis but showed extensive splenic involvement with stage IV disease. Alternatively, the morphologic findings in this case were like to those seen in other Primary Splenic Diffuse Large B Cell Lymphoma reported in the literature. In conclusion, the present report emphasizes the need for additional case studies and research on the biology of this unusual subset of lymphoma.

EZB-type diffuse large B-cell lymphoma cell lines have superior migration capabilities compared to MCD-type.

Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent aggressive B-cell lymphoma. The group is heterogeneous and the outcome is variable. A variety of approaches have been employed with the objective of improving the stratification of DLBCL patients according to their prognosis, based on the cell of origin. Recently, distinct genetic subtypes of DLBCL have been identified. Given the importance of cell migration in immune cells, the objective of this study was to ascertain whether different genetic subtypes of DLBCL exhibit disparate migration abilities. MCD- and EZB-type DLBCL cell lines were subjected to testing to ascertain their basal velocity in straight microchannels and their ability to overcome tight constrictions of 2 μm. The EZB-type cell lines showed superior basal migration velocity and constriction passage time, and a similar trend was observed in live cell imaging of native human DLBCL tissue. In addition, MCD-type DLBCL exhibited significantly elevated levels of nuclear lamin A/C, which is responsible for the stiffness of the nuclear envelope and could thus explain the disparate migration behaviours observed among these subtypes. Our study suggests that different genetic subtypes of DLBCL may not only influence the outcome after therapy but also the motility of the tumour cells.

Rituximab induces ferroptosis and RSL3 overcomes rituximab resistance in diffuse large B-cell lymphoma cells

Rituximab induces ferroptosis and RSL3 overcomes rituximab resistance in diffuse large B-cell lymphoma cells

Integrated multi-omics profiling reveals the ZZZ3/CD70 axis is a super-enhancer-driven regulator of diffuse large B-cell lymphoma cell-natural killer cell interactions.

Tumor immune microenvironment is crucial for diffuse large B-cell lymphoma (DLBCL) development. However, the mechanisms by which super-enhancers (SEs) regulate the interactions between DLBCL cells and tumor-infiltrating immune cells remains largely unknown. This study aimed to investigate the role of SE-controlled genes in regulating the interactions between DLBCL cells and tumor-infiltrating immune cells. Single-cell RNA-seq, bulk RNA-seq and H3K27ac ChIP-seq data were downloaded from the Heidelberg Open Research Data database and Gene Expression Omnibus database. HOMER algorithm and Seurat package in R were used for bioinformatics analysis. Cell proliferation and lactate dehydrogenase (LDH) release was detected by MTS and LDH release assays, respectively. Interaction between B cell cluster and CD8+ T cell and NK cell cluster was most obviously enhanced in DLBCL, with CD70-CD27, MIF-CD74/CXCR2 complex, MIF-CD74/CD44 complex and CCL3-CCR5 interactions were significantly increased. NK cell sub-cluster showed the strongest interaction with B cell cluster. ZZZ3 upregulated the transcription of CD70 by binding to its SE. Silencing CD70 in DOHH2 cells significantly promoted the proliferation of co-cultured NK92 cells and LDH release from DOHH2 cells, which was counteracted by ZZZ3 overexpression in DOHH2 cells. CD70 silencing combined with PD-L1 blockade promoted LDH release from DOHH2 cells co-cultured with NK92 cells. In conclusion, DLBCL cells inhibited the proliferation and killing of infiltrating NK cells by regulating ZZZ3/CD70 axis. Targeting ZZZ3/CD70 axis combined with PD-L1 blockade is expected to be a promising strategy for DLBCL treatment.

Autologous stem cell transplantation in T-cell/histiocyte-rich large B-cell lymphoma: EBMT Lymphoma Working Party study

AbstractAlthough broadly used, consolidative autologous hematopoietic stem cell transplantation (auto-HCT) for relapsed/refractory (R/R) T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) has never been specifically investigated. Here, we have analyzed outcomes of auto-HCT for THRLBCL compared with diffuse large cell B-cell lymphoma not otherwise specified (DLBCL). Eligible for this retrospective registry study were adult patients with R/R THRLBCL and DLBCL, respectively, who underwent a first auto-HCT in a salvage-sensitive disease status as assessed by positron emission tomography–computed tomography between 2016 and 2021 and were registered with the European Society for Blood and Marrow Transplantation database. The primary end point was progression-free survival (PFS) 2 years after transplantation. A total of 201 patients with THRLBCL and 5543 with DLBCL were included. There were no significant differences in terms of disease status at HCT, pretreatment lines, and interval from diagnosis to transplant between the cohorts, but patients with THRLBCL were significantly younger, contained a higher proportion of men, and had a better performance status. Compared with DLBCL, THRLBCL was associated with significantly better 2-year PFS (78% vs 59%; P < .001) and overall survival (OS, 81% vs 74%; P = .02) because of a significantly lower 2-year relapse incidence (16% vs 35%; P < .001). On multivariate analysis, favorable relapse risk (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.31-0.7) and PFS (HR, 0.58; 95% CI, 0.41-0.82) of patients with THRLBCL remained significant, whereas OS benefits (HR, 0.78; 95% CI, 0.54-1.12) did not. These results were validated in a propensity score–matched analysis. These data prove auto-HCT as an effective treatment option for salvage-sensitive R/R THRLBCL.

Diffuse Primary Anaplastic Large Cell Lymphoma of the Lung Diagnosed by Transbronchial Lung Biopsy Under Severe Respiratory Failure

Diffuse Primary Anaplastic Large Cell Lymphoma of the Lung Diagnosed by Transbronchial Lung Biopsy Under Severe Respiratory Failure

Comprehensive Bioinformatic Investigation of TP53 Dysregulation in Diverse Cancer Landscapes.

P53 overexpression plays a critical role in cancer pathogenesis by disrupting the intricate regulation of cellular proliferation. Despite its firmly established function as a tumor suppressor, elevated p53 levels can paradoxically contribute to tumorigenesis, influenced by factors such as exposure to carcinogens, genetic mutations, and viral infections. This phenomenon is observed across a spectrum of cancer types, including bladder (BLCA), ovarian (OV), cervical (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and uterine corpus endometrial carcinoma (UCEC). This broad spectrum of cancers is often associated with increased aggressiveness and recurrence risk. Effective therapeutic strategies targeting tumors with p53 overexpression require a comprehensive approach, integrating targeted interventions aimed at the p53 gene with conventional modalities such as chemotherapy, radiation therapy, and targeted drugs. In this extensive study, we present a detailed analysis shedding light on the multifaceted role of TP53 across various cancers, with a specific emphasis on its impact on disease-free survival (DFS). Leveraging data from the TCGA database and the GTEx dataset, along with GEPIA, UALCAN, and STRING, we identify TP53 overexpression as a significant prognostic indicator, notably pronounced in prostate adenocarcinoma (PRAD). Supported by compelling statistical significance (p < 0.05), our analysis reveals the distinct influence of TP53 overexpression on DFS outcomes in PRAD. Additionally, graphical representations of overall survival (OS) underscore the notable disparity in OS duration between tumors exhibiting elevated TP53 expression (depicted by the red line) and those with lower TP53 levels (indicated by the blue line). The hazard ratio (HR) further emphasizes the profound impact of TP53 on overall survival. Moreover, our investigation delves into the intricate TP53 protein network, unveiling genes exhibiting robust positive correlations with TP53 expression across 13 out of 27 cancers. Remarkably, negative correlations emerge with pivotal tumor suppressor genes. This network analysis elucidates critical proteins, including SIRT1, CBP, p300, ATM, DAXX, HSP 90-alpha, Mdm2, RPA70, 14-3-3 protein sigma, p53, and ASPP2, pivotal in regulating cell cycle dynamics, DNA damage response, and transcriptional regulation. Our study underscores the paramount importance of deciphering TP53 dynamics in cancer, providing invaluable insights into tumor behavior, disease-free survival, and potential therapeutic avenues.

Identification of a novel form of caspase-independent cell death triggered by BH3-mimetics in diffuse large B-cell lymphoma cell lines

BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.

Effects of polyphyllin Ⅶ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

Artesunate induces ferroptosis by regulating MT1G and has an additive effect with doxorubicin in diffuse large B-cell lymphoma cells

Diffuse Large B-cell lymphoma (DLBCL) is a highly aggressive disease with heterogeneous outcomes and marked variability in the response to chemotherapy. DLBCL comprises two major subtypes: germinal centre B-cell-like (GCB) and activated B-cell-like (ABC). Our study highlights the extensive antitumour activity of artesunate (ART) against both major DLBCL subtypes. Transcriptome analysis suggests the potential involvement of ferroptosis in artesunate-induced cell death. Because of low glutathione (GSH) and glutathione peroxidase 4 (GPX4) levels, along with the accumulation of free iron (Fe2+), artesunate induces the excessive production of reactive oxygen species (ROS), ultimately leading to ferroptosis, a form of cell death driven by phospholipid peroxidation. A putative target of artesunate, metallothionein 1G (MT1G), was selected for further analysis. Subsequent studies revealed that inhibiting MT1G expression in vitro significantly impedes the ferroptosis-promoting activity of artesunate by reducing lipid peroxidation and iron accumulation. We also showed that the combination of artesunate and doxorubicin had a marked additive inhibitory effect on GCB and ABC DLBCL cells. In conclusion, artesunate induces ferroptotic death in GCB and ABC DLBCL cells by attenuating the GPX4/GSH antioxidant defence system and increasing intracellular iron levels, indicating its therapeutic potential for relapsed or refractory DLBCL.

Abstract 6069: Discovery and preclinical development of orally bioavailable potent BTK degrader, and its early clinical development for the treatment of relapsed and refractory B-cell malignancies

Abstract Background : Bruton tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade, which is critical for growth and survival of B-cell lymphoma or leukemia. BTK inhibitors have shown anti-tumor activity in patients with B-NHL. However, nearly half of the patients on current BTK inhibitors discontinue the treatment due to adverse effects and/or mutation-induced drug resistance. A degrader is a heterobifunctional chemical compound aimed at eliminating disease-causing proteins, rather than simply inhibiting proteins’ enzymatic function, through the ubiquitin-proteasome system (UPS)-mediated protein degradation. This approach offers a unique solution for addressing relapse or refractory disease by removing overexpressed and mutated proteins, unlike traditional inhibitors. We thus designed UBX-382, a degrader targeting BTK. Results : UBX-382 showed superior degradation activity for both wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of half-maximal degradation concentration when compared to the competing degrader programs in diffuse large B-cell lymphoma cell line. UBX-382 effectively addressed 7 out of 8 previously reported resistant BTK mutants in in vitro experiments and outperformed ibrutinib, ARQ-531, and MT-802 in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK-expressing TMD-8 cells. Remarkably, oral dosing of UBX-382 for &amp;lt;2 weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. Following this, further optimization was carried out for potency and PK profile. We nominated the candidate for IND filing and are planning a phase 1 clinical trial. For the first-in-human, open-label, multicenter, phase 1 study, patients in part 1 (dose escalation with BOIN design) had R/R B-cell malignancies and had received at least 2 prior therapies. The candidate will be administered orally at 5 mg once daily followed by 2 days, for a 7-day schedule over 28-day cycles. Part 2 (dose expansion) consisted of disease-specific cohorts, including CLL/SLL, DLBCL, MCL, FL, MZL, and WM. The primary endpoints were safety and tolerability, along with the definition of the maximum tolerated dose. Secondary endpoints included pharmacokinetics/pharmacodynamics and preliminary efficacy. Conclusions : We have discovered potent BTK degrader exhibiting excellent anti-tumor activity in vitro and in vivo. In pre-clinical safety studies, the candidate of UBX demonstrated an acceptable safety profile. The candidate of UBX is currently preparing phase 1 clinical trials for hematological malignancies. Citation Format: Song Hee Lee, Sun-Mi Yoo, Ye Seul Lim, Han Wool Kim, Je Ho Ryu, Jong Hwan Lim. Discovery and preclinical development of orally bioavailable potent BTK degrader, and its early clinical development for the treatment of relapsed and refractory B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6069.

Abstract 7001: Profiling the CpG methylation patterns of Epstein-Barr virus DNA in lymphoma patients from Ethiopia

Abstract Introduction: Epstein-Barr virus (EBV) is a tumor virus associated with various malignancies, driven by genetic mutations and epigenetic alterations, including DNA methylation. Abnormal CpG methylation holds promise as a diagnostic and therapeutic biomarker for EBV-associated tumors. However, the understanding of EBV's epigenetic mechanisms remain limited. Determining the methylation profile of EBV is crucial for gaining insights into the pathogenesis of EBV-associated tumors, developing diagnostic biomarkers, and exploring potential therapeutic strategies. Therefore, our study aimed to assess the CpG methylation profile of EBV DNA in lymphoma patients from Ethiopia. Methods: This study enrolled 115 lymphoma patients from Tikur Anbessa Specialized Hospital in Addis Ababa, Ethiopia. Plasma and formalin-fixed paraffin-embedded blocks were collected from the participants, and isolation of DNA was performed from these samples. The methylation analysis was performed using the methylation-iPLEX assay. iPLEX capture and extension primers were designed to amplify and target EBV CpGs from bisulfite-converted DNA sequences. Ethical approval was also obtained from respected institutions. Results: Among the 115 participants, 65.2% (n = 75) were male patients. The largest proportion (n = 35, 30.4%) fell within the 41-60 age group. Non-Hodgkin lymphoma (NHL) was diagnosed in 96 patients, while 19 had Hodgkin lymphoma (HL). In the NHL group, the majority (n = 27, 28.1%) were classified as diffuse large cell lymphoma, followed by follicular lymphoma (n = 24, 25%). Out of the different EBV genome methylation sites, we have identified 24 EBV genes to assess the methylation status of the EBV genome. Average methylation levels of these genes were ranged from 45% to 50%. Notably, the HL group exhibited significant heterogeneity in methylation levels, while the CpG methylation status of EBV genes in NHL subtypes demonstrated a consistent pattern. A significant proportion of our samples exhibited methylation loss in the lytic gene BZLF1 and at the origin of replication associated with lytic replication. Conclusions: This study presents the first data from East Africa, shedding light on the EBV DNA methylation status in a diverse group of lymphoma patients. Further investigations in different locations are necessary to elucidate the impact of EBV DNA methylation on tumor progression. Citation Format: Seifegebriel T. Feleke, Kidist Z. Shita, Abdulaziz A. Sherif, Fisihatsion T. Woldegebriel, Christoph Weigel, Elshafa H. Ahmed, Tamrat A. Zeleke, Robert A. Baiocchi. Profiling the CpG methylation patterns of Epstein-Barr virus DNA in lymphoma patients from Ethiopia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7001.

Kaempferol inhibits the proliferation and migration of Epstein-Barr virus-positive diffuse large B-cell lymphoma cells

Purpose: To assess the effect of kaempferol on the growth, apoptosis, and motility of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) cells, and to elucidate its mechanism of action. &#x0D; Methods: Human DLBCL cells were used to generate EBV-positive (EBV +) cell line Pfeiffer. CCK-8, colony formation, flow cytometry (FCM), and immunoblot assays were employed to determine the effects of kaempferol on the growth and apoptosis of EBV + DLBCL cells. Cell motility was evaluated by Transwell and immunoblot assays. Immunoblot assay was further conducted to unravel the mechanism of action. &#x0D; Results: Kaempferol inhibited the growth of EBV + DLBCL cells. It also enhanced the apoptosis of EBV + DLBCL cells, but inhibited the motility of the cells. Furthermore, kaempferol inhibited PI3K/AKT axis, thereby suppressing DLBCL. &#x0D; Conclusion: Kaempferol inhibits the growth and motility of EBV + DLBCL cells via PI3K/AKT axis. It is therefore a potential drug for the treatment of DLBCL.

Lymphoma’s last therapeutic option: Freeze it!

Cryoablation has been used for removal of solid cancers in selected settings. We describe its application in a patient who was diagnosed with diffuse large cell lymphoma. A residual lymphoma lesion involving the right adrenal gland, still PET-positive after multiple lines of chemotherapy, was eradicated with the use of cryoablation. The patient is alive and in complete response at last follow-up, over 4 years from the procedure. Our data suggest that cryoablation may have a role also in the setting of lymphoma.

Statement of Retraction: Knockdown of zinc finger protein 267 suppresses diffuse large B-cell lymphoma progression, metastasis, and cancer stem cell properties

Statement of Retraction: Knockdown of zinc finger protein 267 suppresses diffuse large B-cell lymphoma progression, metastasis, and cancer stem cell properties

CD30 protects EBV-positive diffuse large B-cell lymphoma cells against mitochondrial dysfunction through BNIP3-mediated mitophagy

Epstein-Barr virus (EBV) positive diffuse large B-cell lymphoma (EBV+ DLBCL) predicts poor prognosis and CD30 expression aggravates the worse consequences. Here, we reported that CD30 positivity was an independent prognostic indicator in EBV+ DLBCL patients in a retrospective cohort study. We harnessed CRISPR/Cas9 editing to engineer the first loss-of-function models of CD30 deficiency to identify that CD30 was critical for EBV+ DLBCL growth and survival. We established a pathway that EBV infection mediated CD30 expression through EBV-encoded latent membrane protein 1 (LMP1), which involved NF-κB signaling. CRISPR CD30 knockout significantly repressed BCL2 interacting protein 3 (BNIP3) expression and co-IP assay indicated a binding between CD30 and BNIP3. Moreover, silencing of CD30 induced mitochondrial dysfunction and suppressed mitophagy, resulting in the accumulation of damaged mitochondria by depressing BNIP3 expression. Additionally, CRISPR BNIP3 knockout caused proliferation defects and increased sensitivity to apoptosis. All the findings reveal a strong relationship between mitophagy and adverse prognosis of EBV+ DLBCL and discover a new regulatory mechanism of BNIP3-mediated mitophagy, which may help develop effective treatment regimens with anti-CD30 antibody brentuximab vedotin to improve the prognosis of CD30+ EBV+ DLBCL patients.

MYCT1 inhibits hematopoiesis in diffuse large B-cell lymphoma by suppressing RUNX1 transcription

BackgroundThe abnormality of chromosomal karyotype is one factor causing poor prognosis of lymphoma. In the analysis of abnormal karyotype of lymphoma patients, three smallest overlap regions were found, in which MYCT1 was located. MYCT1 is the first tumor suppressor gene cloned by our research team, but its studies relating to the occurrence and development of lymphoma have not been reported.MethodsR banding analyses were employed to screen the abnormality of chromosomal karyotype in clinical specimen and MYCT1 over-expression cell lines. FISH was to monitor MYCT1 copy number aberration. RT-PCR and Western blot were to detect the mRNA and protein levels of the MYCT1 and RUNX1 genes, respectively. The MYCT1 and RUNX1 protein levels in clinical specimen were evaluated by immunohistochemical DAB staining. The interaction between MYCT1 and MAX proteins was identified via Co-IP and IF. The binding of MAX on the promoter of the RUNX1 gene was detected by ChIP and Dual-luciferase reporter assay, respectively. Flow cytometry and CCK-8 assay were to explore the effects of MYCT1 and RUNX1 on the cell cycle and proliferation, respectively.ResultsMYCT1 was located in one of three smallest overlap regions of diffuse large B-cell lymphoma, it altered chromosomal instability of diffuse large B-cell lymphoma cells. MYCT1 negatively correlated with RUNX1 in lymphoma tissues of the patients. MAX directly promoted the RUNX1 gene transcription by binding to its promoter region. MYCT1 may represses RUNX1 transcription by binding MAX in diffuse large B-cell lymphoma cells. MYCT1 binding to MAX probably suppressed RUNX1 transcription, leading to the inhibition of proliferation and cell cycle of the diffuse large B-cell lymphoma cells.ConclusionThis study finds that there is a MYCT1-MAX-RUNX1 signaling pathway in diffuse large B-cell lymphoma. And the study provides clues and basis for the in-depth studies of MYCT1 in the diagnosis, treatment and prognosis of lymphoma.

Comprehensive analysis and immunohistochemistry localization of NRP1 expression in pancancer and normal individual tissues in relation to SARS‑CoV‑2 susceptibility.

Neuropilin 1 (NRP1/CD304) is a typical membrane-bound co-receptor for vascular endothelial growth factor, semaphorin family members and viral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, NRP1 expression levels across cancer types and the potential role of SARS-CoV-2 infection in patients with cancer are not clear. Online databases, such as The Cancer Genome Atlas database of Human Protein Atlas, Gene Expression Profiling Interactive Analysis and cBioPortal were used for the expression analysis in this study. Immunohistochemical (IHC) staining for NRP1 was performed in the tissues of patients with non-small cell carcinoma. As a result, it was found that NRP1 mRNA and protein expression levels were highest in the female reproductive tissues and the respiratory system, specifically in the nasopharynx, bronchus and fallopian tube, as well as in adipocytes, hepatic stellate cells, Sertoli cells, endothelial cells and dendritic cells. IHC showed that the NRP1 protein was mainly localized to the cytoplasm and membrane in the tissues of patients with non-small cell carcinoma, demonstrating its role in lung infection by SARS-CoV-2, due to invasion of cell membranes by the virus. Levels of NRP1 mRNA were significantly increased in lymphoid neoplasm diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma (KIRC), pancreatic adenocarcinoma, stomach adenocarcinoma and thymoma, and significantly decreased in cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney chromophobe, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, uterine corpus endometrial carcinoma and uterine carcinosarcoma, compared with corresponding healthy tissues in pancancer, indicating roles for viral invasion in most cancer types. Moreover, low NRP1 expression was significantly associated with long overall survival (OS) time in adrenocortical carcinoma, brain lower grade glioma, stomach adenocarcinoma and uveal melanoma, but with short OS time in KIRC only. The ENST00000374867.6 (NRP1-202) isoform is most highly expressed in most cancer types and thus could be involved in tumorigenesis and SARS-CoV-2 invasion in cancer patients. NRP1 may be involved in SARS-CoV-2 invasion in patients with cancer, including those with lung cancer.

Abstract B170: The in vitro anti-tumor activity of the multi-kinase inhibitor nemtabrutinib (ARQ-531, MK-1026) is seen across multiple B-cell lymphoma subtypes, only partially overlapping with what achieved by single BTK inhibition

Abstract Background. Nemtabrutinib is a reversible, ATP competitive tyrosine kinase inhibitor of BTK and of several other kinases, including LYN and MEK1 (Reiff et al, Cancer Discov 2018). Nemtabrutinib has shown preclinical and clinical activity in chronic lymphocytic leukemia (CLL), also against tumors bearing mutations affecting the C481 amino acid of BTK (Reiff et al, Cancer Discov 2018; Eradat et al, ICML 2023), which is commonly mutated in patients treated with 1st and 2nd generation inhibitors such as ibrutinib, acalabrutinib, zanubrutinib (Thompson &amp; Tam, Blood 2023). Based on these data, a phase 3 trial is comparing the drug against chemo-immunotherapy in untreated-CLL patients (NCT05624554). Phase 1/2 studies are currently exploring nemtabrutinib in various B-cell lymphomas as single-agent (NCT05673460, NCT04728893) and combined with the ROR1-targeting antibody drug conjugate zilovertamab vedotin (NCT05458297). Due to the ability of nemtabrutinib to block multiple kinases, we exposed a large series of lymphoma cell lines to nemtabrutinib to understand the lymphomas that might most benefit. Methods. Nemtabrutinib (MedChemExpress) was assessed for its anti-proliferative activity at 72h across 54 cell lines, comprising germinal-center B-cell like (GCB) DLBCL (n.=18), activated B-cell like (ABC) DLBCL (n.=8), mantle cell lymphoma (MCL) (n.=10), marginal zone lymphoma (MZL) (n.=6), CLL (n.=2), plus other two derived from B- and eight from T-cell (n.=8) lymphomas. Results. The median IC50 for nemtabrutinib was 1.4 µM (95%CI, 0.8-2.3 µM) across all 54 lymphoma cell lines tested. Higher activity was found in B (median-IC50, 1.1 µM) compared to T (median IC50, 22.6 µM) cell lymphomas. Strong anti-lymphoma activity was observed in CLL cell lines (median IC50, 389 nM), MCL (median IC50, 627 nM), in one primary mediastinal B cell lymphoma (815 nM) and one canine diffuse large B-cell lymphoma cell line (389 nM). No difference was seen between ABC (median IC50, 1 µM) and GCB (median IC50, 1.2 µM) DLBCL. In DLBCL, nemtabrutinib anti-tumor activity was not affected by the presence of inactive TP53, BCL2 and/or MYC translocations. Taking advantage of publicly available ibrutinib data, we observed that nemtabrutinib and the 1st generation BTK inhibitor behaved similarly across 45 lymphoma cell lines (Pearson correlation r=0.5, P=0.001), especially in MCL (r=0.8, P=0.005) and ABC-DLBCL (r=0.75, P=0.039), but not in GCB-DLBCL, in which only nemtabrutinib was active. Further analyses integrating baseline transcriptome and mutational data are underway. Conclusions. Nemtabrutinib showed anti-tumor activity across B-cell lymphomas, higher in some histotypes (including MCL, CLL). The pattern of anti-tumor activity was only partially overlapping by what achieved by the BTK inhibitor ibrutinib: while MCL and ABC-DLBCL responded similarly, activity in GCB-DLBCL was seen only with nemtabrutinib. Citation Format: Giulio Sartori, Filippo Spriano, Luciano Cascione, Chiara Tarantelli, Alberto J. Arribas, Luca Aresu, Davide Rossi, Giovanna Damia, Massimo Broggini, Francesco Bertoni. The in vitro anti-tumor activity of the multi-kinase inhibitor nemtabrutinib (ARQ-531, MK-1026) is seen across multiple B-cell lymphoma subtypes, only partially overlapping with what achieved by single BTK inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B170.

Determinants of Response to Anti CD-19 CAR-T Cells for Diffuse Large B-Cell Lymphoma in Pre-Treatment Peripheral Blood Mononuclear Cells Using Single-Cell RNA-Seq

Background: Chimeric antigen receptor T (CAR-T) cell therapy has become the standard of care in patients with relapsed/refractory diffuse large cell lymphoma (R/R DLBCL), providing durable remission in 40% of patients. Lack of response is likely to be attributed to inter- and intra-patient variability, including PBMC heterogeneity and apheresis material composition, CAR-T infusion product, in-vivo expansion and activity of CAR-T cells and the development of tumor-intrinsic mechanisms. Current methods for evaluating CAR-T cell activation and potency have limited predictive value for clinical outcomes. To address this challenge, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) obtained from patients with R/R LBCL patients at the time of apheresis for the manufacturing of anti-CD19 CAR-T cell therapy. Methods: Between 10/2020 and 03/2022, 31 patients who underwent lymphopheresis were enrolled. Samples were obtained - 1) At day of lymphopheresis from peripheral blood; 2) On the day of infusion of CAR-T from the product; and 3) On day +7 after infusion from peripheral blood. All patients had day +30 PET-CT. In addition, PBMCs samples from healthy donors were used as control to analyze baseline characteristics compared to the DLBCL patients. PBMCs from patients of healthy donors were purified from frozen blood samples by density gradient separation, stained using human CD45 antibody and loaded onto a 10x Chromium system. libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit. The data was processed and analyzed using the Seurat R package. The study was approved by the local Ethic committee. Results: Patient characteristics: 31 RR DLBCL patients (42% females; median age 72, range 47-81 years), all treated with CAR-T cells therapy as third line of therapy, participated in the study. All patients underwent lymphopheresis, followed by CAR-T production. Disease status at lymphodepletion was complete remission (CR, n=4, 13%), partial remission (n=6, 19%), and progressive disease (n=21, 68%) and patients received either tisagenlecleucel (n=20, 65%) or axicabtagene ciloleucel (n=11, 35%) . PET scan performed on day 30 post treatment revealed that 68% and 32% obtained CR, and PD, respectively. scRNA analysis revealed differences in PBMC composition between healthy donors and LBCL patients (Figure 1A), with lower levels of naïve and memory T cells and elevated levels of activated monocytes in LBCL patients, suggesting a shift towards an inflamed and pre-dysfunctional immune state. Categorization of LBCL patients into responders (PR/CR on day +30 PET-CT) and non-responders (PD on day +30 PET-CT) revealed differences in immune cell subsets between responders and non-responders (Figure 2A), with enriched inflammation-related pathways in non-responders, In contrast, responders exhibited a distinct healthy-like immune profiles, particularly high levels of naive T cells, similar to that obtained in their age matched healthy controls. Of note, non-responders showed alterations in immune cell subsets, including increased levels of T regulatory cells and effector NK cells. Furthermore, non-responders demonstrated a prominent inflammatory immune profile, characterized by gene pathways related to inflammation and activation, such as type I and III IFN-G, and TNFb response (Figure 1B). Notably, an IFN-G score was significantly higher in non-responder's monocytes subpopulations, compared to responders. Conclusions: These findings indicate that the success of CAR-T therapy could be determined by the molecular characteristics and subpopulation distribution of each patient's PBMC. The diverse immune profiles of DLBCL patients might impact the effectiveness of their immune cells in generating successful CAR-T products or providing a conducive environment for CAR T cells to combat cancer. Furthermore, these findings emphasize the potential of scRNA-seq in evaluating PBMC from CAR-T patients prior to treatment, allowing the potential calculation of an “immune potency score” associated with the probability of a positive outcome from CAR-T treatment of DLBCL patients. This score could potentially be used by oncologists in decision-making processes to determine the appropriate candidates for CAR-T therapy.