B-cell Lines Research Articles

Abstract A26: SOX11 drives B1a lineage commitment and B1a/MCL-like lymphoma formation in mice

Abstract Mantle cell lymphoma (MCL) is a highly aggressive B cell lymphoma that accounts for 6% of all non-Hodgkin lymphomas and is characterized by abnormal proliferation of mature antigen- naive B lymphocytes. Despite recent advances in patient risk stratification, MCL remains incurable due to frequent relapses and resistance to therapy. SOX11 is a key transcription factor in the pathogenesis of MCL and is highly expressed in conventional MCL, but it is not expressed in the normal B cells or in the indolent leukemic nonnodal MCL subtype. Although SOX11 was identified as crucial oncogene that supports maintenance of MCL, its role in the initiation of MCL remains largely unknown. Here, we developed a novel mouse model for condition overexpression of SOX11 and a eGFP reporter and found that elevated levels of SOX11 in B-cells are sufficient to drive MCL-like lymphomas in mice. Moreover, SOX11 expression synergized with loss of p53 and overexpression of Ccnd2 to form aggressive MCL-like CD19+CD5+CD23−IgM+IgD+ lymphomas, with a median survival of 314 (p53Mb1 ; n=11), 165 (SOX11/p53Mb1 ; n=19), and 134 (SOX11/Ccnd2/p53Mb1 ; n=15) days. These SOX11-driven lymphomas show transcriptional, immunophenotypic and functional similarities with both murine B1a cells and MCL patients. SOX11 overexpression skews B-cell development towards the B1a and marginal zone B-cell lineage and it block B2-cell development at the Pro-B and Pre-B cell stage, which coincides with onset eGFP that is a measure of expression of Cre and SOX11. In contrast to cyclin D2-driven MCL-like model, SOX11 is inducing a pre-lymphoma stage, in which there is a progressive accumulation of B1a cells in peripheral blood, peritoneal cavity, spleen and bone-marrow. The pre-lymphoma B1a cells have a BCR repertoire which was biased towards binding of self-antigens, such as phosphatidylcholine, and have increased BCR signaling activity. To prove that embryonic-derived B1a cells were the cell-of-origin of SOX11-driven MCL-like lymphoma, we transplanted CD19+ B-cells from SOX11/p53Mb1 fetal livers into secondary recipients and found that these embryonic cells could give rise to MCL-like lymphomas. Next, we wanted to determine how SOX11 is inducing the pre-lymphoma phase, either by expanding existing B1a cells, having a developmental bias towards B1a cells, or by the ability to reprogram B2-cells towards a B1a cell fate. Therefore, we crossed SOX11/p53 mice with a tamoxifen-inducible CreERT2 line and transplanted different sorted B1 and B2 subsets after a short tamoxifen induction ex vivo in immunocompromised mice. We found that SOX11 OE can redirect mature B2 cells to form B1a/MCL lymphomas. Overall, SOX11 is a key B1a lineage factor in mice and its aberrant expression is leading to the formation of MCL-like lymphomas in mice. Citation Format: Tim Pieters, Steven Goossens, Pieter Van Vlierberghe. SOX11 drives B1a lineage commitment and B1a/MCL-like lymphoma formation in mice [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A26.

Increased serum level of alpha-2 macroglobulin and its production by B-lymphocytes in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), the most common adult’s leukemia in the western world, is caused in 95% of the cases by uncontrolled proliferation of monoclonal B-lymphocytes. The complement system in CLL is chronically activated at a low level via the classical pathway (CP). This chronic activation is induced by IgG-hexamers, which are formed after binding to alpha-2-macroglobulin (A2M). The study investigated for the first time the serum levels of A2M in CLL patients, their association with the disease severity, and A2M production by the malignant B-lymphocytes. Blood samples were collected from 65 CLL patients and 30 normal controls (NC) subjects, and used for quantifications of the A2M levels, the complement activation marker (sC5b-9), the complement components C2, C3 and C4, and clinical biochemistry and hematology parameters. The production of A2M was studied in B-lymphocytes isolated from blood samples as well as in CLL and non-CLL cell lines.The serum A2M levels were significantly higher in CLL patients vs NCs, showing values of 3.62 ± 0.22 and 1.97 ± 0.10 mg/ml, respectively. Within the CLL group, A2M levels correlated significantly with the disease stage, with sC5b-9, and with clinical indicators of the disease severity. Increased A2M production was showed in three out of four CLL B-lymphocytic lines that were studied, as compared to non-CLL lines, to a non-lymphocytic line, and to blood-derived primary B-lymphocytes. A2M production was further increased both in primary cells and in the CLL cell-line after incubation with CLL sera, compared to NC sera. This study shows for the first time that serum A2M levels in CLL are significantly increased, likely due to A2M production by the malignant B-lymphocytes, and are correlated with the disease severity and with chronic complement activation. The moderate change in A2M production after incubation with NC sera in-vitro supports the hypothesis that inhibition of excess A2M production can be achieved, and that this may potentially down-regulate the IgG-hexamerization and the resulting chronic CP activation. This may also help restore complement system activity, and eventually improve complement activity and immunotherapy outcomes in CLL.

Clinical and Cytogenetic Characteristics of Children With Leukemia 20-Year Retrospective Study.

Acute leukemias are the most common malignant diseases in childhood. The aims of this retrospective cohort study were to investigate the frequency of cytogenetic abnormalities in acute pediatric leukemia; the correlation between cytogenetic abnormalities and 5-year survival; and the correlation between cytogenetic abnormalities and clinical and laboratory features. We included 105 patients; acute lymphoblastic leukemia (ALL) had 80.9% patients, B-cell lineage ALL (B-ALL) 84.7% of them, and T-cell lineage (T-ALL) 15.3%. The overall 5-year survival for B-ALL was 85.9% and for T-ALL was 84.6%. The most common cytogenetic abnormalities in patients with B-ALL were t(12;21)(p13.2;q22.1); ETV6-RUNX1 with 22.2% and hyperdiploidy with 19.4%. Our survival analysis showed that t(12;21)(p13.2;q22.1); ETV6-RUNX1 and t(1;19)(q23;p13.3); TCF3-PBX1 had the best 5-year survival with 100% of patients surviving, whereas t(v;11q23.3); KMT2A rearranged had the worst 5-year survival of just 33.3% of patients surviving after 5 years. We found no difference in 5-year survival in B-ALL when comparing clinical features. Acute myelogenous leukemia had 20 patients with 70.6% 5-year survival. The most common cytogenetic abnormality in acute myelogenous leukemia was t(8;21)(q21;q22.1); RUNX1-RUNX1T1 (20%). In conclusion, this study showed the correlation of different cytogenetic abnormalities with 5-year survival in B-ALL patients. Such correlation was not found when comparing clinical features and 5-year survival of patients with B-ALL. This emphasized the significance of cytogenetic analysis in pediatric leukemia.

Adaptation of cell spreading to varying fibronectin densities and topographies is facilitated by β1 integrins.

Cells mechanical behaviour in physiological environments is mediated by interactions with the extracellular matrix (ECM). In particular, cells can adapt their shape according to the availability of ECM proteins, e.g., fibronectin (FN). Several in vitro experiments usually simulate the ECM by functionalizing the surfaces on which cells grow with FN. However, the mechanisms underlying cell spreading on non-uniformly FN-coated two-dimensional substrates are not clarified yet. In this work, we studied cell spreading on variously functionalized substrates: FN was either uniformly distributed or selectively patterned on flat surfaces, to show that A549, BRL, B16 and NIH 3T3 cell lines are able to sense the overall FN binding sites independently of their spatial arrangement. Instead, only the total amount of available FN influences cells spreading area, which positively correlates to the FN density. Immunocytochemical analysis showed that β1 integrin subunits are mainly responsible for this behaviour, as further confirmed by spreading experiments with β1-deficient cells. In the latter case, indeed, cells areas do not show a dependency on the amount of available FN on the substrates. Therefore, we envision for β1 a predominant role in cells for sensing the number of ECM ligands with respect to other focal adhesion proteins.

Clonal germinal center B cells function as a niche for T-cell lymphoma

AbstractAngioimmunoblastic T-cell lymphoma (AITL) is proposed to be initiated by age-related clonal hematopoiesis (ACH) with TET2 mutations, whereas the G17V RHOA mutation in immature cells with TET2 mutations promotes the development of T follicular helper (TFH)-like tumor cells. Here, we investigated the mechanism by which TET2-mutant immune cells enable AITL development using mouse models and human samples. Among the 2 mouse models, mice lacking Tet2 in all the blood cells (Mx-Cre × Tet2flox/flox × G17V RHOA transgenic mice) spontaneously developed AITL for approximately up to a year, while mice lacking Tet2 only in the T cells (Cd4-Cre × Tet2flox/flox × G17V RHOA transgenic mice) did not. Therefore, Tet2-deficient immune cells function as a niche for AITL development. Single-cell RNA-sequencing (scRNA-seq) of >50 000 cells from mouse and human AITL samples revealed significant expansion of aberrant B cells, exhibiting properties of activating light zone (LZ)-like and proliferative dark zone (DZ)-like germinal center B (GCB) cells. The GCB cells in AITL clonally evolved with recurrent mutations in genes related to core histones. In silico network analysis using scRNA-seq data identified Cd40–Cd40lg as a possible mediator of GCB and tumor cell cluster interactions. Treatment of AITL model mice with anti-Cd40lg inhibitory antibody prolonged survival. The genes expressed in aberrantly expanded GCB cells in murine tumors were also broadly expressed in the B-lineage cells of TET2-mutant human AITL. Therefore, ACH-derived GCB cells could undergo independent clonal evolution and support the tumorigenesis in AITL via the CD40–CD40LG axis.

The long non-coding RNA CRNDE promotes osteosarcoma proliferation and migration by sponging miR-136-5p/MRP9 axis.

BackgroundThe long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene has been found to be upregulated in several solid tumors. Whether CRNDE affects osteosarcoma (OS) and its underling mechanism remains unknown.MethodsTumor tissues and corresponding normal tissues were collected from 45 patients with OS. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied to determine lncRNA CRNDE level in the tissues. Participants were divided into a high CRNDE group and a low CRNDE group according to the median value of lncRNA CRNDE expression detected by in situ hybridization (ISH). The differences between high and low expression of lncRNA CRNDE in patients were compared clinically by chi-square test. Kaplan-Meier survival analysis was applied to analyze the relationship between lncRNA CRNDE expression and patient survival. Subsequently, silencing or overexpression of lncRNA CRNDE were performed in MG63 and 143B cell lines, qRT-PCR was applied to verify the expression of lncRNA CRNDE, miR-136-5p, and MRP9; dual-luciferase reporter assay was used to evaluate the targeting relationship between miR-136-5p, lncRNA CRNDE, and Cell Counting Kit-8 (CCK8), wound-healing, and Transwell assays were used to analyze for cell proliferation, migration, and invasion, respectively, and western blot was used to detect expression in cells.ResultsThe expression of CRNDE in OS tissues was higher than that in normal tissues. High lncRNA CRNDE expression was significantly associated with clinical stage, lung metastasis, and poor prognosis in OS patients. Additionally, overexpression of lncRNA CRNDE promoted proliferation and migration of OS cells. Bioinformatics analysis showed that lncRNA CRNDE competitively inhibited miR-136-5p through acting as a competitive endogenous RNA (ceRNA). It was also revealed that miR-136-5p is a binding target gene of lncRNA CRNDE and that MRP9 is involved in this process as a downstream target gene of miR-136-5p.ConclusionsThe lncRNA CRNDE promotes the proliferation and migration of OS cells by regulating the miR-136-5p/MRP9 pathway, and lncRNA CRNDE can be a significant marker of OS prognosis.

Chemical composition and biological activities of essential oils from six lamiaceae folk medicinal plants.

Essential oils have attracted wide attention in recent years due to their extensive applications in natural functional ingredients, pharmaceutical preparations, biomedical products, and the cosmetics industry. In this study, the chemical compositions and biological activities of essential oils extracted from six Lamiaceae herbs, including Pogostemon cablin (Blanco) Benth. (PCEO), Perilla frutescens (L.) Britton (PFEO), Salvia japonica Thunb. (SJEO), Rosmarinus officinalis L. (ROEO), Lavandula angustifolia Mill. (LAEO), and Agastache rugosa (Fisch. & C. A. Mey.) Kuntze (AREO), were determined and analyzed. A total of 167 components were identified from the six essential oils by GC-MS analysis, with 35, 24, 47, 46, 54, and 37 components in PCEO, PFEO, SJEO, ROEO, LAEO, and AREO, respectively. Hierarchical cluster analysis of chemical compositions showed that the composition of the six essential oils was significantly different in content, and they were clearly divided into six classes. However, all of these six essential oils exhibited promising anti-inflammatory activity by inhibiting the expression of interleukin-1, interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 in rats with adjuvant arthritis, among which PFEO had the best performance. In addition, the six essential oils displayed significant cytotoxicity on B16 (IC50 = 86.91–228.91 μg/mL) and LNCaP cell lines (IC50 = 116.4–189.63 μg/mL). Meanwhile, all of them presented satisfactory antioxidant activity (IC50 = 4.88–13.89 μg/mL) compared with Trolox C (IC50 = 13.83 μg/mL), and SJEO (IC50 = 7.93 μg/mL) served as an optimal candidate natural antioxidant by DPPH assay. Taken together, these results indicate that the six Lamiaceae essential oils manifest excellent and diverse biological activities, enabling them to be used as perfect natural functional ingredients in antioxidant, antitumor, or anti-arthritic drugs. This study provides more references for pharmaphylogeny research and drug discovery from folk medicinal plants.

Commonly Assessed Markers in Childhood BCP-ALL Diagnostic Panels and Their Association with Genetic Aberrations and Outcome Prediction.

Immunophenotypic characterization of leukemic cells with the use of flow cytometry (FC) is a fundamental tool in acute lymphoblastic leukemia (ALL) diagnostics. A variety of genetic aberrations underlie specific B-cell precursor ALL (BCP-ALL) subtypes and their identification is of great importance for risk group stratification. These aberrations include: ETV6::RUNX1 fusion gene, Philadelphia chromosome (BCR::ABL1 fusion gene), rearrangements of the KMT2A, TCF3::PBX1 fusion gene and changes in chromosome number (hyperdiploidy and hypodiploidy). Diagnostic panels for BCP-ALL usually include B-cell lineage specific antigens: CD19, CD10, CD20, maturation stage markers: CD34, CD10, CD38, TdT, IgM and other markers useful for possible genetic subtype indication. Some genetic features of leukemic cells (blasts) are associated with expression of certain antigens. This review comprehensively summarizes all known research data on genotype-immunophenotype correlations in BCP-ALL. In some cases, single molecules are predictive of particular genetic subtypes, i.e., NG2 with KMT2A gene rearrangements or CD123 with hyperdiploidy. However, much more information on possible genotype or prognosis can be obtained with wider (≥8-color) panels. In several studies, a quantitative antigen expression scale and advanced statistical analyses were used to further increase the specificity and sensitivity of genotype/immunophenotype correlation detection. Fast detection of possible genotype/immunophenotype correlations makes multicolor flow cytometry an essential tool for initial leukemia diagnostics and stratification.

A De-novo Case of Philadelphia Chromosome Positive B-Cell Acute Lymphoblastic Leukaemia in Elderly with Past History of Endometrial Carcinoma

Introduction: Philadelphia chromosome is well-known chromosomal abnormality in Chronic Myeloid Leukaemia (CML). However, B-acute lymphoblastic leukemia (B-ALL) with Philadelphia-positive (Ph′+) is a neoplasm of lymphoblast committed to the B-cell lineage, occurs in about 20 % to 30 % of all cases in adults. The incidence increases with age, up to 50 % of ALL diagnosed in individual’s ≥50 years old. The clinical presentation of B-ALL Ph′+ is similar to B-ALL but is more common in adults than in children. Case Report: A 64 year-old female patient presented to Medicine OPD with complaints of fever, weakness and uneasiness, along with old history of endometrial carcinoma. She was further investigated and diagnosed as De-novo case of B-precursor ALL with positive BCR-ABL fusion gene as no chemotherapy was given for previously reported malignancy. Conclusion: Here, we like to emphasize that overlapping symptoms may lead to delay in diagnosis. Hence, clinician should always investigate the patient thoroughly, in order to diagnose the patient on time. So that, treatment can be started as early as possible to avoid fatal outcomes in Ph′ Chromosome positive B cell ALL, as it has worst prognosis.

A Novel Anti-CD22 scFv.Bim Fusion Protein Effectively Induces Apoptosis in Malignant B cells and Promotes Cytotoxicity.

CD22 is a B-cell surface antigen which is highly expressed in cancerous B-cell lineages. Anti-CD22 antibodies are currently under focus as promising biologics against hematologic B-cell malignancies. Herein, we introduce a novel active recombinant anti-CD22 scFv.Bim fusion protein for targeting this cancerous antigen. An expression cassette encoding anti-CD22 scFv.Bim fusion protein was expressed in Pichia pastoris. The binding ability, cytotoxicity, and apoptotic activity of the purified recombinant protein against CD22+ Raji cell line were assessed by flow cytometry, microscopy, and MTT assay. Using bioinformatics, the 3D structure of the fusion protein and its interaction with CD22 were assessed. The in vitro binding analysis by immunofluorescence microscopy and flow cytometry demonstrated the specific binding of scFv.Bim to CD22+ Raji cells but not to CD22- Jurkat cells. MTT data and Annexin V/PI flow cytometry analysis confirmed the apoptotic activity of anti-CD22 scFv.Bim against Raji cells but not Jurkat cells. In silico analysis also revealed the satisfactory stereochemical quality of the 3D model and molecular interactions toward CD22. This novel recombinant anti-CD22 scFv.Bim fusion protein could successfully deliver the pro-apoptotic peptide, BIM, to the target cells and thus nominates it as a promising molecule in treating B-cell malignancies.

Formation of Lymphoma Hybrid Spheroids and Drug Testing in Real Time with the Use of Fluorescence Optical Tweezers.

Interactions between stromal and lymphoma cells in the bone marrow are closely related to drug resistance and therapy failure. Physiologically relevant pre-clinical three-dimensional (3D) models recapitulating lymphoma microenvironmental complexity do not currently exist. In this study, we proposed a scheme for optically controlled hybrid lymphoma spheroid formation with the use of optical tweezers (OT). Following the preparation of stromal spheroids using agarose hydrogel, two aggressive non-Hodgkin lymphoma B-cell lines, Ri-1 (DLBCL) and Raji (Burkitt lymphoma), were used to conduct multi-cellular spheroid formation driven by in-house-developed fluorescence optical tweezers. Importantly, the newly formed hybrid spheroid preserved the 3D architecture for the next 24 h. Our model was successfully used for the evaluation of the influence of the anticancer agents doxorubicin (DOX), ibrutinib (IBR), and AMD3100 (plerixafor) on the adhesive properties of lymphoma cells. Importantly, our study revealed that a co-treatment of DOX and IBR with AMD3100 affects the adhesion of B-NHL lymphoma cells.

Eryngium billardieri Extract and Fractions Induce Apoptosis in Cancerous Cells.

Eryngium is a genus flowering plant in the Umbelliferae family, having pharmacological properties, such as anti-inflammatory and anti-diabetic. Given the nature of melanoma and breast cancers in recent years and the fact that the anti-cancer properties of Eryngium billardieri on mentioned cell lines have not been studied, the present study was conducted to explore these properties. The mechanisms of cytotoxicity and apoptosis of aerial parts of various extracts and fractions of E. billardieri on cancerous cells and normal cells were investigated. Samples were collected from natural habitats, dried and then extracted by Soxhlet apparatus with solvents of n-Hex, DCM and methanol, respectively. The cytotoxic effects of the extracts were investigated by the MTT method on MCF7, B16 and HFF-2 classes for 24 and 48 hours. Flow cytometry was also used to investigate the mechanism of cytotoxicity, and it has been confirmed by Real-time PCR of p53 and Bax genes as to which comprise the apoptosis regulatory proteins. Meanwhile, volatile compounds of extracts were identified by the GC-MS method. The obtained data showed that the n-Hex extract of E. billardieri on B-16 and MCF7 cell lines and dichromethane extract on MCF7 cell line had the most significant cytotoxic effect compared to DMSO control (p-value <0.001). Our finding showed that the mechanism of n-Hex extract with 80% and 100% vlc fractions on B16 induced apoptotis compared to HFF-2 control cells; moreover, n-Hex extract and 80% vlc fraction on MCF7 were apoptotic. The major compounds of n-Hex, DCM, and 80% and 100% fractions of n-Hex extract obtained from GC-MS were non-terpenoid. Non-terpenoid compounds of E. billardieri can be responsible for exhibiting the most cytotoxic effects on MCF7 and B16 cell lines with apoptotic mechanism, and n-Hex extract was found to have the most significant inhibitory effect on cancerous cells compared to the HFF-2 control cells by employing the mechanism of apoptosis.

Enhancement of antiproliferative potential of metformin against melanoma mice B16 cells using an optimized liposomal drug delivery system.

Metformin-loaded liposomes were optimized for enhanced antiproliferative activity against melanoma. Box-Behnken design and response surface methodology were employed to optimize entrapment efficiency, ex-vivo permeation and vesicle size. The optimized formulation was prepared by both the lipid hydration method and the modified injection method for comparison. Different concentrations of Pluronic F127 were employed for modification. Selected Pluronic-modified formulation (lipid molar concentration 55 mmol, cholesterol 30% and drug loading 52.9 mg) was characterized for morphology, entrapment efficiency, permeation and vesicle size. The optimized formulation resulted in entrapment efficiency of 41.7 ± 0.01%, vesicle size of 1.405 ± 0.061 µm and percentage of permeation was 67 ± 5.5%. The improved cytotoxic effect of the selected formulation against melanoma mice B16 cell line compared with metformin solution was determined using MTT assay. Compared with the corresponding drug solution, the Pluronic-modified optimized liposomes displayed a highly efficient cytotoxic effect as evidenced by significant lowering in IC50 -887.3 ± 23.2 and 26.71 ± 0.69 μg/ml, respectively, P < 0.0001. This study introduces an optimized liposomal formulation with enhanced cytotoxic effect against melanoma B16 cell line.

COVID-19 and Cancer: Special Considerations for Patients Receiving Immunotherapy and Immunosuppressive Cancer Therapies.

Patients with cancer generally have a higher risk of adverse outcomes from COVID-19, with higher age, male sex, poor performance status, cancer type, and uncontrolled malignant disease as the main risk factors. However, the influence of specific cancer therapies varies and raises concerns during the pandemic. In patients undergoing cancer immunotherapy or other immunosuppressive cancer treatments, we summarize the evidence on outcomes from COVID-19; address the safety, immunogenicity, and efficacy of COVID-19 vaccination; and review COVID-19 antiviral therapeutics for the patient with cancer. Despite higher mortality for patients with cancer, treatment with immune checkpoint inhibitors does not seem to increase mortality risk based on observational evidence. Inhibitory therapies directed toward B-cell lineages, including monoclonal antibodies against CD20 and CAR T-cell therapies, are associated with poor outcomes in COVID-19; however, the data are sparse. Regarding vaccination in patients receiving immune checkpoint inhibitors, clinical efficacy comparable to that in the general population can be expected. In patients undergoing B-cell-depleting therapy, immunogenicity and clinical efficacy are curtailed, but vaccination is not futile, which is thought to be due to the cellular response. Vaccine reactogenicity and toxicity in all groups of patients with cancer are comparable to that of the general population. Preexposure prophylaxis with monoclonal antibodies directed against the viral spike may provide passive immunity for those not likely to mount an adequate vaccine response. If infected, prompt treatment with monoclonal antibodies or oral small molecule antivirals is beneficial, though with oral antiviral therapies, care must be taken to avoid drug interactions in patients with cancer.

Photodynamic Antitumor Activity of 5,15‐Bis(perfluorophenyl)‐10‐(4‐carboxyphenyl)corrole and its Gallium(III) and Phosphorus(V) Complexes

This work reports the preparation and characterization of an A2 B corrole 5,15-bis(perfluorophenyl)-10-(4-carboxyphenyl)corrole and its gallium(III) and phosphorus(V) complexes. Their in-vitro photodynamic anticancer activities against A549, MDA-MB-231, B16, HepG2, and Hela cell lines were also investigated. Among three compounds, phosphorus(V) complexexhibits the best photostability, highest fluorescence quantum yields (ΦF =0.138), and the highest singlet-oxygen quantum yields (ΦΔ =0.87). Also, the phosphorus(V) complex exhibits the best photodynamic antitumor activity against MDA-MB-231 cells with a low IC50 (0.08 μM) upon light irradiation at 625±2 nm, which is much lower than commercial PDT drug Temoporfin (0.1 μM) at the same conditions. The cellular localization assay confirmed that the phosphorus(V) complexis mainly distributed in the cytoplasm and have a good ability to produce reactive oxygen species (ROS) under light illumination, which would further cause oxidative damage to tumor cells and finally result in the apoptosis. After PDT treatment, phosphorus(V) complex may cause tumor cell arrest at the G2/M stage. The preliminary results showed phosphorus(V) corrole complex is a good candidate for photodynamic therapy (PDT) of tumors.

Primary Intestinal Lymphoma in Rabbits

Neoplasms of the intestinal tract are uncommon in rabbits and primary lymphoma of the intestinal tract is rare. This case series is the first detailed description of primary intestinal lymphoma in rabbits. We reviewed four cases of primary intestinal lymphoma in rabbits aged 5-9.5 years old with an average age of 7.8 years. Neoplastic cells in three cases were large (8μm diameter) while one case had intermediate cells (5μm diameter). Neoplastic lymphocytes were of B-cell lineage and characterized by intense, multifocal, membranous immunoreactivity for CD79a and no immunoreactivity to CD3. Based on the Revised European-American Classification of Lymphoid Neoplasms/World Health Organization classification, three of the cases were consistent with diffuse large B-cell lymphoma and the case with intermediate-sized neoplastic cells was consistent with lymphoblastic lymphoma.

P1596: PREDIAGNOSTIC ANTIMICROBIAL USE IN CLL, MM, FL AND DLBCL

Background: Chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and multiple myeloma (MM) are B-cell lineage malignancies all sharing an immune dysfunction leading to an increased risk of infection. CLL, FL and MM are preceded by indolent precursor states; more specifically monoclonal B-cell lymphocytosis (MBL), in situ follicular neoplasia (ISFN), and monoclonal gammopathy of uncertain significance (MGUS), respectively. MBL, ISFN and MGUS may be detected decades prior to overt malignancy. Although CLL and FL may transform into DLBCL, evidence for a premalignant DLBCL precursor is scarce. We have previously demonstrated an increased use of antibiotics up to 20 years prior to CLL diagnosis (Andersen et al. Leukemia. 2020). Whether patients with DLBCL, FL or MM share the same trait as patients with CLL remains unknown. Aims: To assess use of antimicrobials for patients with FL, DLBCL and MM prior to diagnosis Methods: We included all patients diagnosed with CLL since 2008, small lymphocytic lymphoma (SLL), DLBCL, FL and symptomatic MM since 2005 through Danish national patient registries (DCLLR, LYFO and DaMyDa; https://www.rkkp.dk/). We analyzed patients with CLL and SLL jointly, as they are recognized as the same disease entity and treated similarly. Likewise, patients with FL grade 3b are clinically similar to DLBCL, thus patients with FL treated with R-CHOP-like regimens within 3 months of diagnosis were considered grade 3b and analyzed together with DLBCL. Data on antimicrobial (AM) prescriptions (Rx) were retrieved from the Danish national database for reimbursed prescriptions (DNDRP) using WHO ATC codes. To characterize the use of prediagnostic AM use, we included AM Rx from January 2004 until date of diagnosis. Patients with multiple malignancies were classified according to the first occurring diagnosis. Statistical analyses were performed using R software. Results: We followed 21,822 patients for a mean of 9.8 person-years prior to diagnosis (SD+/- 4.8), from which a total of 138,805 AM Rx were identified for 18,840 (86.3%) distinct patients. Within the cohort, 6377 (29.2%) were diagnosed with CLL accounting for 42,107 (30.33%) AM Rx, 5803 (26.6%) were diagnosed with MM accounting for 38,417 (27.7%) AM Rx, 6735 (30.9%) were diagnosed with DLBCL accounting for 41,623 (30.0%) AM Rx, and 2907 (13.3%) were diagnosed with FL accounting for 16,658 (12.0%) AM Rx. Thus, 2982 (13.7%) patients had no Rx prior to diagnosis of whom 989 (33.2%) had DLBCL, 775 (26.0%) had CLL, 722 (24.2%) had MM, and 496 (16.6%) had FL. Stratified by disease, patients later diagnosed with CLL and MM accounted for more AM Rx than patients later diagnosed with DLBCL and FL (45.0% vs 41.6% vs 37.8% vs 35.4% with >3 AM Rx, respectively; Table 1). In particular, more penicillin and macrolide were prescribed prior to diagnosis of CLL and MM compared to patients later diagnosed with DLBCL and FL. Image:Summary/Conclusion: These preliminary results indicate a higher rate of infections, as assessed by number of AM Rx, in individuals later developing CLL and MM compared to individuals later developing DLBCL and FL. Thus, MM and CLL seems to be prone to immune dysfunction also prior to diagnosis. Whether immune dysfunction plays a role in development of the disease or is due to the premalignant MBL and MGUS warrants further investigations.

Atypical B cells in chronic infectious diseases and systemic autoimmunity: puzzles with many missing pieces.

The world’s struggle to contain the SARS-CoV-2 epidemic, primarily through vaccination, has highlighted the importance of better understanding the biology of B cells that participate in defense against infectious diseases, both acute and chronic. Here, we focus on a population of human B cells, termed atypical B cells (ABCs), that comprise a distinct B-cell lineage that differentiates from naive B cells in an interferon-γ-driven process, and are infrequent in healthy individuals but significantly expanded in chronic infectious diseases, including malaria, as well as in systemic autoimmune diseases such as systemic lupus erythematosus (SLE). Recent comparisons of ABCs by single-cell RNAseq provided evidence that ABCs in diverse chronic infectious diseases and in systemic autoimmune diseases are highly related and share common drivers of differentiation and expansion. However, ABCs in different diseases are not identical and also show discrete disease-specific features. Here, we compare and contrast key features of two ABC populations, namely those that are expanded in individuals living in malaria-endemic areas of the world versus those in SLE patients. This comparison is of interest as it appears that unique features of these two diseases result in participation of autoreactive ABCs in parasite-specific responses in malaria but in pathogenic autoimmune responses in SLE. A better understanding of the commonality and differences in the ABC responses in these two diseases may provide critical insights into the development of vaccines that drive pathogen-specific antibody responses and avoid autoimmunity.

New Triazacycloalkane Derivatives as Cytotoxic Agents for CLL Treatment: From Proof of Concept to the Targeting Biomolecule.

The 1,4,7-tris-(2-pyridinylmethyl)-1,4,7-triazacyclononane ligand (no3py) and its bifunctional analogue no3pyCOOK were synthesized to investigate their action toward zinc(II) depletion related to the apoptosis phenomenon in chronic lymphocytic leukemia (CLL) cells. no3py was used as the "free" ligand, while its "graftable" derivative was conjugated on a newly synthesized bifunctional sialoglycan, 6'-SL-NH2, selected to specifically bind CD22 biomarker expressed on the B-CLL cell surface. Both compounds were produced with good yields thanks to a Sonogashira coupling reaction and an orthoester function, respectively, for the chelator and the targeting moiety. The newly reported bioconjugate 6'-SL-no3py was then obtained through a peptidic coupling reaction. Biological in vitro studies of no3py and 6'-SL-no3py consisting of real-time detection of cell health (cytotoxicity and proliferation) and caspases 3/7 activation (crucial enzymes whose activation triggers cell death signaling pathways) have been investigated. First, Ramos, Daudi, and Raji B-cell lines, which present different sensitivity to zinc(II) content variation, were incubated with no3py and 6'-SL-no3py. Then, a videomicroscope allowed the real-time monitoring of the morphological changes leading to cell death from the detection of the cytotoxicity, the antiproliferative effect, and the caspasic activity. In terms of mechanism, the Zn2+ chelator cytotoxic effect of no3py has been evidenced by a culture medium ion supplementation study and by the decrease of intracellular fluorescence of Zn-specific fluorophore zinquin in the presence of no3py and 6'-SL-no3py chelators. Finally, flow cytometry analysis with classical Annexin V staining was conducted to detect no3py- and 6'-SL-no3py-induced apoptotic cell death in B-CLL cells. Time-course analysis, using the Incucyte Live-Cell Analysis System, demonstrated that no3py induced cell death in a time- and dose-dependent manner with variability across cell lines. 6'-SL-no3py exhibited the same dose-dependent trend as no3py, showing the efficiency of the targeting moiety. In both cases, the chelators depicted proliferation curves that were inversely correlated with kinetic death. Morphological changes specific to apoptosis and caspase 3/7 activation were observed for the three cell lines treated with no3py and 6'-SL-no3py, highlighting their role as apoptotic agents. A higher concentration of 6'-SL-no3py is needed to reach 50% of the B-CLL mortality, confirming a targeting of the chelator to the cell membrane. Overall, our results proved that the biological properties of the triazamacrocyclic chelator still remain even after addition of the targeting moiety. The free chelator as well as the bioconjugate constitute promising cytotoxic agents for CLL therapy through apoptosis induction.

Abstract 5371: Repurposing erbb2 (HER2/neu) as a neoantigen target for neu-negative tumors using a novel erbb2-encoding lentivirus

Abstract INTRODUCTION: HER2/neu is a potent biomarker for which anti-HER2/neu antibodies offer significant responses. However, many cancers, such as melanoma, have less than 5% expression of HER2/neu. The purpose of this study was to determine whether induced expression of erbb2 (mouse HER2/neu analogue) could function as a neoantigen target for anti-erbb2 antibody. METHODS: RT-PCR was used to construct a rat erbb2-encoding vector using the live, non-replicating lentivirus pLenti6.3. erbb2-encoding pLenti6.3 was transfected in vitro into the B16 mouse melanoma cell line, which has less than 5% endogenous expression of erbb2. The generated erbb2+ B16 cell line was designated B16/neu. Anti-erbb2 antibody (7.16.4) was used to treat C57Bl/6 male and female mice bearing B16/neu (200 ug intraperitoneally three times per week for 3 weeks). To confirm activity of 7.16.4, this antibody (100 ug per T75 flask) was co-cultured with B16/neu in vitro, and cell growth/confluence was measured. RESULTS: The novel B16/neu cell line expressed approximately 50% erbb2 in vitro on flow cytometry analysis, compared to 2% errb2 expression in naïve B16. Erbb2/neu expression was confirmed on fluorescent Western blot. Anti-erbb2 antibody (7.16.4) slowed the growth of B16/neu in vitro, confirming the suppressive effects of 7.16.4. In B16/neu-bearing mice, 7.16.4 resulted in effective control of tumor growth and a complete tumor response rate of 35.7%, (5/14 mice) compared to placebo controls (p &amp;lt; 0.01). Interestingly, B16/neu tumors demonstrated slower growth kinetics compared to naïve B16 tumors (p = 0.013), suggesting immune recognition of erbb2 in naïve C57Bl/6 mice as a neoantigen target. No adverse reactions were observed with 7.16.4. In vivo inoculation of erbb2-encoding pLenti6.3 in naïve B16 tumors and treatment of in vivo transfected B16/neu tumors with 7.16.4 comprise ongoing experiments. CONCLUSIONS: erbb2 expression on B16 melanoma was successfully achieved using a novel erbb2-encoding lentivirus. Induced erbb2 served as a neoantigen target for anti-erbb2 antibody. This preclinical study provides translational evidence that induced HER2/neu expression may be utilized to repurpose anti-HER2 antibodies for human malignancies that do not normally express high levels of HER2/neu. Citation Format: Emmanuel M. Gabriel, Brian Necela, Deborah Bahr, Jamie Kaplan, Kristopher Attwood, Keith Knutson. Repurposing erbb2 (HER2/neu) as a neoantigen target for neu-negative tumors using a novel erbb2-encoding lentivirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5371.