B-chronic Lymphocytic Leukemia Research Articles

Histotopography of highly glycosylated lymphocytes and stromal cells in lymph nodes of patients with B-chronic lymphocytic leukaemia

Histotopography of highly glycosylated lymphocytes and stromal cells in lymph nodes of patients with B-chronic lymphocytic leukaemia

Inflammation mediated angiogenesis in chronic lymphocytic leukemia.

Chronic inflammation has been identified in leukemias as an essential regulator of angiogenesis. B-chronic lymphocytic leukemia (CLL) cells secrete high levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 alpha (HIF1α). The aim was to assess the role of inflammation in activation of angiogenic factors: endothelial nitric oxide synthase (eNOS), HIF1α and VEGF via proliferation related signaling pathways and VEGF autocrine control. We isolated mononuclear cells (MNC) and CD19+ cells from peripheral blood of 60 patients with CLL. MNC were treated with pro-inflammatory interleukin-6 (IL-6) and VEGF, in combination with inhibitors of JAK1/2 (Ruxolitinib), mTOR (Rapamycin), NF-κB (JSH23), SMAD (LDN-193189) and PI3K/AKT (Ly294002) signaling pathways, to evaluate eNOS, VEGF and HIF1α expression by immunoblotting, immunocytochemistry and RT-qPCR. Also, we investigated IL-6 dependent neovascularization in human microvascular endothelial cells (HMEC-1) in co-culture with MNC of CLL. The angiogenic factors eNOS, VEGF and HIF1α had significantly higher frequencies in MNC of CLL in comparison to healthy controls (p < 0.001) and CD19+ cells of CLL. IL-6 increased the quantity of HIF1α (p < 0.05) and VEGF positive cells in the presence of JSH23 (p < 0.01). VEGF increased HIF1α (p < 0.05), and decreased eNOS gene expression (p < 0.01) in MNC of CLL. VEGF significantly (p < 0.001) increased the number of HIF1α positive MNC of CLL, prevented by inhibitors of JAK1/2, PI3K and mTOR signaling pathways. VEGF stimulation of SMAD (p < 0.05) and STAT5 (p < 0.01) signaling has been prevented by inhibitors of JAK1/2, mTOR, PI3K and SMAD signaling, individually (p < 0.01) or mutually (p < 0.001). Also, we showed that MNC of CLL and IL-6 individually stimulate neovascularization in co-culture with HMEC-1, without a cumulative effect. We demonstrated elevated angiogenic factors in CLL, while VEGF and IL-6 independently stimulated HIF1α. VEGF stimulation of HIF1α was mostly mTOR dependent, while IL-6 stimulation was NF-κB dependent.

Progressive multifocal leukoencephalopathy in a patient with B-cell chronic lymphocytic leukemia after COVID-19 vaccination, complicated with COVID-19 and mucormycosis: a case report

BackgroundProgressive multifocal leukoencephalopathy (PML) is a rare and fatal opportunistic viral demyelinating infectious disease of the central nervous system (CNS). There are various clinical presenting symptoms for the disease.Case presentationThis paper presents a clinical case of PML in a patient with B-Chronic lymphocytic leukemia (B-CLL), previously treated with Chlorambucil, later complicated later with COVID-19 and mucormycosis.ConclusionPML can develop in the setting of cellular immune dysfunction. Late diagnosis of this disease based on nonspecific symptoms is common, therefore when we face a neurological complication in a CLL or immunocompromised patient, we should consider PML infection. A remarkable feature of this case is the possible triggering effect of COVID-19 vaccination for emergence of PML as the disease can be asymptomatic or sub-clinical before diagnosis.

Blocking Orai1 constitutive activity inhibits B-cell cancer migration and synergistically acts with drugs to reduce B-CLL cell survival

Orai1 channel capacity to control store-operated Ca2+ entry (SOCE) and B-cell functions is poorly understood and more specifically in B-cell cancers, including human lymphoma and leukemia. As compared to normal B-cells, Orai1 is overexpressed in B-chronic lymphocytic leukemia (B-CLL) and contributes in resting B-CLL to mediate an elevated basal Ca2+ level through a constitutive Ca2+ entry, and in BCR-activated B-cell to regulate the Ca2+ signaling response. Such observations were confirmed in human B-cell lymphoma and leukemia lines, including RAMOS, JOK-1, MEC-1 and JVM-3 cells. Next, the use of pharmacological Orai1 inhibitors (GSK-7975 A and Synta66) blocks constitutive Ca2+ entry and in turn affects B-cell cancer (primary and cell lines) survival and migration, controls cell cycle, and induces apoptosis through a mitochondrial and caspase-3 independent pathway. Finally, the added value of Orai1 inhibitors in combination with B-CLL drugs (ibrutinib, idelalisib, rituximab, and venetoclax) on B-CLL survival was tested, showing an additive/synergistic effect including in the B-cell cancer lines. To conclude, this study highlights the pathophysiological role of the Ca2+ channel Orai1 in B-cell cancers, and pave the way for the use of ORAI1 modulators as a plausible therapeutic strategy.

The role of polymorphic cytochrome P450 gene (CYP2B6) in B-chronic lymphocytic leukemia (B-CLL) incidence and outcome among Egyptian patients.

Cytochromes P450 (CYPs) play a prominent role in catalyzing phase I xenobiotic biotransformation and account for about 75% of the total metabolism of commercially available drugs, including chemotherapeutics. The gene expression and enzyme activity of CYPs are variable between individuals, which subsequently leads to different patterns of susceptibility to carcinogenesis by genotoxic xenobiotics, as well as differences in the efficacy and toxicity of clinically used drugs. This research aimed to examine the presence of the CYP2B6*9 polymorphism and its possible association with the incidence of B-CLL in Egyptian patients, as well as the clinical outcome after receiving cyclophosphamide chemotherapy. DNA was isolated from whole blood samples of 100 de novo B-CLL cases and also from 100 sex- and age-matched healthy individuals. The presence of the CYP2B6*9 (G516T) polymorphism was examined by PCR-based allele specific amplification (ASA). Patients were further indicated for receiving chemotherapy, and then they were followed up. The CYP2B6*9 variant indicated a statistically significant higher risk of B-CLL under different genetic models, comprising allelic (T-allele vs. G-allele, OR = 4.8, p < 0.001) and dominant (GT + TT vs. GG, OR = 5.4, p < 0.001) models. Following cyclophosphamide chemotherapy, we found that the patients with variant genotypes (GT + TT) were less likely to achieve remission compared to those with the wild-type genotype (GG), with a response percentage of (37.5% vs. 83%, respectively). In conclusion, our findings showed that the CYP2B6*9 (G516T) polymorphism is associated with B-CLL susceptibility among Egyptian patients. This variant greatly affected the clinical outcome and can serve as a good therapeutic marker in predicting response to cyclophosphamide treatment.

ROR1 expression in mature B lymphoid neoplasms by flow cytometry.

Immunophenotyping by flow cytometry is an integral part of the diagnosis and classification of leukemias/lymphomas. The expression of ROR1 associated with chronic B lymphocytic leukemia (CLL) is well described in the literature, both in its diagnosis and in the follow-up of minimal residual disease (MRD) research, however, there are few studies regarding the expression pattern of ROR1 in other subtypes of mature B lymphoid neoplasms. With the aim of evaluating the expression of ROR1 and associating it with the expression of other important markers for the differentiation of mature B lymphoid neoplasms (MBLN), 767 samples of cases that entered our laboratory for immunophenotyping with clinical suspicion of MBLN were studied. ROR1 expression is predominant in CD5+/CD10- neoplasms. Overall, positive ROR1 expression was observed in 461 (60.1%) cases. The CD5+/CD10- group had a significantly higher proportion of ROR1 positive samples (89.9%) and more brightly expressed ROR1 than the other groups. Our results highlight the importance of evaluating ROR1 expression in the diagnosis of MBLN to contribute to the differential diagnosis, and possibly therapy of mainly CLL, and indicate that this marker could be considered as a useful addition to immunophenotypic panels, particularly for more challenging cases.

Association of CYP1A1 T3801C (rs4646903) variant with the susceptibility and progression of B-chronic lymphocytic Leukemia (B-CLL) in the Egyptian population

BackgroundThe frequency of hematological malignancies is increasing universally, and over the last few decades, a significant increase in the incidence of B-chronic lymphocytic leukemia (B-CLL) has been observed. Many studies have revealed the involvement of genetic predisposition along with environmental exposure to genotoxic xenobiotics in the leukemogenesis process of B-CLL. CYP1A1 is a vital member of the cytochromes P450 (CYPs) superfamily, which is involved in pro-carcinogens activation into reactive intermediates during phase I xenobiotic biotransformation. AimThis study aimed to determine the possible role of the CYP1A1*2A (T3801C, rs4646903) single nucleotide polymorphism (SNP) as a risk factor for developing B-CLL, as well as the impact of this SNP on the disease progression and the clinical outcome. Patients and MethodsThe study was conducted on 100 patients newly diagnosed with B-CLL, and 100 healthy individuals with matched ages and sex, served as the control group. CYP1A1 (T3801C) genotyping of all patient and control samples was performed using the PCR-based Restriction Fragment Length Polymorphism (RFLP-PCR) method. In addition, serum levels of both IL-6 and TNF-α were estimated by the ELISA technique. ResultsHigher frequencies of the heterozygous carrier (TC) and homozygous variant (CC) genotypes of the CYP1A1 (T3801C) variant were observed in B-CLL patients compared to the controls (P < 0.001 for both). The frequencies of the CYP1A1 (T3801C) variant indicated a significant elevated risk of B-CLL under various genetic models, including allelic (OR = 8.8, P < 0.001) and dominant (OR = 9.3, P < 0.001) models. In addition, the median IL-6 level was significantly higher in patients with (TC) and (CC) genotypes than in patients with (TT) genotype (P = 0.001 and P < 0.001, respectively). Also, the median TNF-α level was significantly higher in patients with (TC) and (CC) genotypes than in patients with (TT) genotype (P < 0.001 for both). ConclusionOur results showed that the CYP1A1*2A (T3801C, rs4646903) SNP increases the susceptibility to B-CLL incidence and is associated with poor disease progression.

In-vitro Modulation of mTOR-HIF-1α Axis by TLR7/8 Agonist (Resiquimod) in B-Chronic Lymphocytic Leukemia

Targeting toll-like receptors (TLRs), via TLR agonists, has been implicated in the regulation of immunometabolism. B-chronic lymphocytic leukemia (B-CLL) represents a suitable model for B-cell derived malignancies with shifted metabolic adaptations. Several signaling pathways have been found to be critical in metabolic reprogramming of CLL, including mechanistic target of rapamycin- hypoxia inducible factor-1α (mTOR- HIF-1α) pathway, the main metabolic regulator of glycolysis. Here, we investigated the effect of TLR7/8 agonist (Resiquimod) on the expression of mTOR and HIF-1α in patients with CLL. B cells were purified using Rosettesep Human B cell Enrichment Cocktail (Stem cell Technologies, Vancouver, BC, Canada#15,024) from peripheral venous blood of CLL patients (n = 20) and healthy individuals (n = 15). Isolated B cells were then cultured in both presence and absence of Resiquimod. Gene expression of mTOR and HIF-1α were assessed using qRT-PCR. Resiquimod significantly decreased mTOR and HIF-1α gene expression in both CLL (p < 0.001and p < 0.001, respectively) and Normal B cells (p = 0.004 and p = 0.001, respectively). Resiquimod may reprogram immunometabolism of malignant B-CLL cells via down-regulation of key glycolytic metabolic actors, mTOR and HIF-1α genes. Accordingly, Resiquimod may be an adjuvant as a therapeutic tool for CLL, which needs to be studied further.

Challenges and opportunities of CAR T-cell therapies for CLL

Chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment landscape of blood cancers. These engineered receptors which endow T cells with antibody-like target cell recognition combined with the typical T cell target cell lysis abilities. Introduced into the clinic in the 2010s, CAR T-cells have shown efficacy in chronic B lymphocytic leukemia (CLL), but a majority of patients do not achieve sustained remission. Here we discuss the current treatment landscape in CLL using small molecules and allogeneic stem cell transplantation, the niche CAR T-cells filled in this context, and what we have learned from biomarker and mechanistic studies. Several product parameters and improvements are introduced as examples of how the bedside-to-bench is translated into improved CAR T-cells for CLL. We hope to convey to our readers the crucial role translational medicine plays in transforming the treatment outcomes for patients with CLL and how this line of research is an essential component of modern medicine.

A Flow Cytometry Panel for Differential Diagnosis of Mantle Cell Lymphoma from Atypical B-Chronic Lymphocytic Leukaemia

Differential diagnosis of chronic lymphoproliferative disorders (CLDs) has remained challenging due to the highly variable morphology features and immunophenotyping. Currently, the development of multiple-marker panel analyses by flow cytometry has opened a broad way for diagnosis of CLDs. We analyzed the peripheral blood and bone marrow samples of 131 patients with B-cell CLDs (including 91 chronic lymphocytic leukemia (CLL), 15 atypical CLL, 14 mantle cell lymphoma (MCL), and 11 CD5-/CD10-lymphoma patients) from April 2018 to April 2019, using a panel of specific markers by flow cytometry. Our results indicated that the expression pattern of CD22, CD23, FMC-7, and CD5 allowed us to accurately and differentially diagnose the B-CLL, MCL, and CD5-/CD10- lymphoma, while it was not capable of differentiating MCL from atypical CLL. We, however, found that the expression patterns of CD38 and immunoglobulin light chain differed significantly between atypical B-CLL and MCL. CD38 and lambda light chain were remarkably expressed in MCL patients (92.8% and 85%, respectively) compared to the atypical CLL (1.1% and 0% respectively), with the p value less than 0.001 for both markers. In contrast to MCL patients, all the patients with atypical CLL, expressed kappa light chain. The immunohistochemistry method used for cyclin D1 confirmed that the flow cytometry detection of kappa and lambda light chains could provide a new approach with high sensitivity (91%) and moderate specificity (50%) to distinguish MCL patients from atypical B-CLL. Expression of CD5, CD20 (bright), CD22, FMC-7, CD38, and lambda light chain with no expression of CD23 can accurately detect MCL and differentiate it from atypical B-CLL

MiRNA profile at diagnosis predicts treatment outcome in patients with B-chronic lymphocytic leukemia: A FILO study.

ClinicalTrials.gov, identifier NCT01370772.

Risk factors for iron metabolic disorders in newly diagnosed B-chronic lymphocytic leukemia patients

Due to the importance of increasing the incidence conditions of B cell chronic lymphocytic leukemia (B-CLL) that requiring accurate knowledge of the biology behind the pathogenesis and risks of this disease of each individual patient, this study was designed to investigate if hormonal alterations such as Hepcidin, Erythropoietin, and oxidant-antioxidant status may be useful in the understanding of B-CLL development in addition the relationship of these parameters with anemia in advance clinical stage in newly diagnosed patients with B-CLL. This study was conducted on 60 newly diagnosed patients with B-CLL and 30 control group .The socio-demographic information including (age and sex) and clinical data were collected for the period from 20 February until 25 December 2021. The result of the present study showed that out of the 60 newly diagnosed B-CLL patients, 39 was male and 21 was females, with the mean age ( 64.04 ± 5.6 ) years and out of the 30 controls group ,12 was males and 18 was females with the mean age of ( 63.33 ± 4.70) years .therefore, it was a higher prevalence p value ≤0.05 of newly diagnosed B-CLL patients in males (65.0 %) compare to females (35.0 % ).

Decreased cell proliferation and induced apoptosis in human B-chronic lymphocytic leukemia following miR-221 inhibition through modulation of p27 expression

BackgroundThis study aimed to investigate the effects of the miR-221 inhibition on the human B-chronic lymphocytic leukemia (B-CLL) cell viability and the p27 gene expression, to introduce a new treatment approach for this type of cancer. In this context, the cyclin-dependent kinase (Cdk) inhibitor 1B (p27Kip1) is considered as an enzyme inhibitor that encodes a protein belonging to the Cip/Kip family of the Cdk inhibitor proteins.MethodsThe affected miR-221 inhibition in the B-CLL cell viability was initially assessed. The inhibition of miR-221 in the B-CLL cell line (183-E95) was thus performed using locked nucleic acid (LNA) as an antagomir. After the LNA-anti-miR-221 transfection, the miR-221 quantification, cell viability, and apoptosis assays were evaluated at different intervals by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and flow cytometry (FC), respectively. The qRT-PCR was also completed for the p27 gene. The data were subsequently analyzed by independent-samples t-test and one-way analysis of variance (ANOVA).ResultsA gradual reduction was observed in the B-CLL cell viability, and consequently the transfected LNA-anti-miR cell viability reached below 55% of the untreated cells after 72 h of transfection. A statistically significant difference was found in the cell viability between the LNA-anti-miR-treated and control groups (p-value ≤ 0.043). The downregulation of miR-221 in the B-CLL (183-E95) cells was further conducted by LNA-anti-miR-221.ConclusionThe miR-221 inhibition significantly decreases cell viability through augmenting the p27 gene expression and inducing apoptosis. Moreover, the findings demonstrated that the inhibition of miR-221 might be a new treatment approach for B-CLL, although more confirmation is needed by investigating appropriate animal models.

Correlation between CD200 expression and oxidant-antioxidant status in newly diagnosed B-chronic lymphocytic leukemia patients

B cell chronic lymphocytic leukemia (B-CLL) is the highest incidence of lymphoproliferative malignant disease in most adults in the western world. Due to the importance of the pathogenicity and risk factors of this disease, this study was designed to analyze the expression the pattern of CD200 by flow cytometer immunophenotyping (FCI) and the association between this marker and some clinical features and oxidant-antioxidant status in B-chronic lymphocytic leukemia patients. This study was conducted on 60 newly diagnostic patients with B-CLL and 30 apparently healthy individuals as control group. The socio-demographic information including (age and sex) and clinical data were also collected in period 20 February 2021 to 25 December. The result of statistical analysis of the present study showed that out of the 60 patients, 39 were male and 21 females, with 30 controls (12 males and 18 females). The mean age of adults patients was (64.04 ± 5.6) years, and the adults control mean age of (63.33 ± 4.70) years therefore, there was a higher prevalence p value ≤0.05 of newly diagnosed CLL patients in males (65.0 %) in compared with females (35.0 %).

Molecular characterization of a Tunisian B-lineage leukemia using Kde gene rearrangements

Background: Molecular approaches applied to the study of B-lineage leukemia have made significant contributions to understanding and to characterizing the disease. The Kde rearrangement was targeted as clonal marker to characterize Tunisian B-chronic lymphocytic leukemia (B-CLL) and B-acute lymphoblastic leukemia (B-ALL). Objective: We used a multiplex PCR to amplify deletional Kde rearrangements and Southern blot assay with Cκ and Kde probes in seven B-CLL and two B-ALL. Direct sequencing was performed in order to determine the n region. Results: An interesting RSS-Kde biallelic rearrangement was found in one adult male patient with B-ALL, since only Vκ-Kde rearrangement has been reported in adult B-ALL. In B-CLL, one case with two clonal cell proliferations in favor of oligoclonality which is very infrequent in B-CLL was identified and might be associated with bad outcome. These first results need more investigation to establish correlation between molecular characteristic and the poor outcome. Conclusion: The molecular approach was successfully improved and applied for clonality assessment of B-lineage leukemia for a better diagnosis and performing monitoring minimal residual disease.

BNT162b2 mRNA COVID-19 Vaccination (BNTCV) in Patients (Pts) Having Hematological Malignancies (HM): Early tolerance with telemedicine application connecting and antibody anti-Spike IgG response (AcAS)

Pts with HM and particularly those under therapy or with secondary immune deficiency have been reported to have a low or delayed specific immune response. In addition, pts receiving rituximab for either HM or auto-immune disease presented a low specific antibody response. Daratumumab, an anti-CD38 moAb was demonstrated to lower normal plasma cells and to reduce polyclonal IgA, M and E in pts with multiple myeloma (MM). It is fundamental to evaluate the early post-vaccination tolerance and the level of seroconversion for such pts.Patients' population.194 pts having HM and followed at the Institute (IC), received at least 2 doses of BNTCV (BioNTech Pfizer, Paris, France) by IM route with 3 weeks between the 2 doses. Currently, analysis was performed on 147 pts, including 63 pts with non-Hodgkin lymphoma (NHL), 18 with B-chronic lymphocytic leukemia (CLL), 34 with MM, 14 with monoclonal gammopathy of undetermined significance (MGUS) and 18 with myeloproliferative disorder (MPD). Mean age was 69 years-old (range 27-92).Follow-up of early tolerance in pts using a telemedicine application.43 pts vaccinated at the IC received Thess®, a telemedicine system connecting the patient to the IC, developed by La Valeriane Inc. (Montpellier, France, www.thess-corp.fr) 24/24h, 7days. Local pain (<1 day) was common and transient, particularly reported after the 2nd dose. Only 4/43 patients reported significant adverse events through telemedicine and followed by a medical call, including mainly severe asthenia for 2 days or more, fever (>38°C) for at least 2 days, headache, or general pain. The satisfaction survey of monitoring system was good. In addition, adherence to vaccination was excellent with only one refusal out of the 194 pts.AcAS follow-upIgG AcAS and IgG+M AcAS were analyzed in the serum 3 to 4 weeks after the 1st dose and 4-8 weeks after the 2 nd dose and every 2 months, respectively by SARS-CoV-2 IgG II Quant ® Assay with a threshold of positivity at 50 AU/mL (Abbott, Rungis, France) and Elecsys ® Anti-SARS-CoV-2 S (Roche Diagnostics, Meylan, France) with a threshold of positivity at 0.8 U/mL. 270 samples were analyzed in duplicate with the 2 assays, by 2 independent labs. Data were reported and statistically analyzed by the clinical research team. 17 results were discordant including 12 with Abbott IgG test undetectable and Roche IgG+M detectable, and 5 with Abbott test detectable and Roche undetectable. After the first dose of BNTCV, 72/147 (49%) pts were seroconverted, including 7/18 (39%) with CLL, 27/63 (43%) with NHL, 10/14 (71%) with MGUS, 17/34 (50%) with MM and 11/18 (61%) with SMD. The median levels of the AcAS response for pts were 0 (range 0-40 000 AU/mL) for the AdviseDX test as compared to 12 normal subjects (mean 257 AU/mL, range 226-283), and 0 U/mL (range 0-2500) for the Elecsys test. 49/75 (65%) of untreated pts were seroconverted after the 1 st dose as compared to 26/72 (36%) of treated patients (p<0.001), with rituximab or ibrutinib as negative factors in addition to low gammaglobulin level (<5g/L, p=0.019), similarly to the IgG level. Analysis of CRP levels, circulating lymphocyte counts, and lymphocyte subpopulations will be performed.After the second dose of BNTCV, 50 pts have currently been tested, with only 25 additional pts seroconverted. The median levels of the AcAS response for pts were 310 AU/mL (range 62-11760) for the AdviseDX test as compared to 12 normal subjects (mean 6725 AU/mL, range 3002-9787), and 1250 U/mL (range 0.87-2500) for the Elecsys test. The 25 patients who were not seroconverted after the 2nd dose received a 3rd dose of BNTCV, including 8 with MM, 9 with NHL, 5 with CLL, 1 with MGUS (with polyneuropathy under daratumumab) and 2 with SMD. Currently, 9 pts were tested after the 3 rd dose. AcAS levels were respectively 268 AU/mL for 1 MM treated by daratumumab, 103 and 313 AU/mL for 2 MM under carfilzomib, 1622 AU/mL for 1 untreated SMD, 29100 AU/mL for 1 untreated MGUS, 537 AU/mL for 1 CLL and 2 negative NHL.In conclusion, there is a need to follow AcAS for pts having HM including SMD after BNTCV aiming to adapt vaccine strategy including a 3 rd dose and eventual recall. As some pts are always negative after a 3 rd dose, vaccination strategy could be discussed by using different combinations of vaccine or the addition of immune adjuvant in a more personalized medicine. In addition, the usage of telemedicine connecting system may help to follow the early tolerance and to improve the pts' adherence. DisclosuresRossi: NPO Petrovax Pharm: Consultancy; LEO Pharma: Consultancy; EUSA Pharma: Consultancy; E-SANA Inc: Other: Co-founder of E-SANA Inc .

Impact of Berberine on the Expression of Apollon Gene in Chronic Lymphocytic Leukemia

Background: The incidence of B-chronic lymphocytic leukemia (B-CLL) resulting from the clonal accumulation of apoptosis-resistant malignant B lymphocytes is growing in the adult population of Iran. Inhibitors of apoptosis proteins (IAPs) are considered as factors that can delay the onset of CLL cell apoptosis. Berberine is an isoquinoline alkaloid isolated from Cotridis rhizoma that exhibits anti-tumor activities through various mechanisms. Objectives: In this study, we investigated the impact of berberine on the level of Apollon expression in peripheral blood mononuclear cells (PBMCs) of 12 cases newly diagnosed with CLL and 6 healthy donors. Methods: At first, the level of Apollon expression was assessed in PBMCs of CLL patients compared to the healthy donors. Peripheral blood mononuclear cells were cultured in RPMI-1640 medium with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin for 48 hours, and the effect of berberine (25 µM) on the level of Apollon expression in CLL patients was assessed and compared to that of healthy donors. Results: We found that the expression level of Apollon was not significantly different between CLL patients and healthy donors (P = 0.640). Moreover, berberine induced no significant differences in Apollon expression as compared to the untreated (control) group (P = 0.545 and P = 0.267 in CLL patients and healthy donors, respectively). Conclusions: Overall, our results suggest that berberine has no direct effect on the expression of Apollon gene in CLL patients, and pro-apoptotic impacts of berberine may be exerted through other mechanisms.

Overcoming of Microenvironment Protection on Primary Chronic Lymphocytic Leukemia Cells after Treatment with BTK and MDM2 Pharmacological Inhibitors.

In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients; however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.

Abstract 915: Preclinical evaluation of anti-CD37 antibody-targeted amanitin conjugates (ATAC) in B-cell malignancies

Abstract Background: ATACs (antibody-targeted amanitin conjugates) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as a toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of ATACs targeting CD37 (member of the tetraspanin family) are presented. CD37 is a transmembrane protein expressed exclusively on cells of the immune system and mainly on mature B-cells, as well as in many B-cell malignancies, including Richter's Syndrome (RS) or Richter´s transformation. RS is a transformation of B cell chronic lymphocytic leukemia, refractory to treatment and carries a poor prognosis, and hence is considered an ideal target for amanitin-based ADCs. Material and methods: Cell lines: CD37-positive: Mec-2 (B-chronic lymphocytic leukaemia); Raji-Luc, Ramos (Burkitt´s lymphoma); CD37-negative: HL-60 (acute lymphocytic leukemia) Antibody: anti-CD37 engineered monoclonal antibody produced at Heidelberg Pharma Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibody. Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega). Animal models: Disseminating Mouse xenograft tumor models (Mec-2 and Raji-Luc) and CD37-positive patient derived xenograft (PDX; Richter's Syndrome) models were performed in single-dose experiments. Tolerability was assessed in non-human primates (NHP). Results: All tested anti-CD37 ATACs showed in vitro cytotoxicity in the picomolar range on CD37-positive cell lines. No cytotoxic activity was observed on CD37-negative cells. In mouse xenograft models, 80 - 100% overall survival was achieved with two anti-CD37 ATACs at doses of 1/16 MTD (Mec-2 model) and 1/64 MTD (Raji Luc Model). Single-dose treatment caused rapid and complete tumor remission already 7 days after treatment. The tested ATACs revealed good tolerability in mice. In NHP, the anti CD37 ATACs revealed good tolerability. Hematology and clinical chemistry parameters were unaffected except liver transaminases and LDH: A mild to moderate and transient increase was observed. Conclusions: Targeted cytotoxic drug delivery to CD37 positive cell lines was achieved by using anti-CD37 ATACs. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in non-human primates. Using ADCs in the therapy of B-cell lymphomas, including malignancies that underwent Richter's transformation, is a promising approach, especially by using a payload whose mode of action differs from other commonly used toxins, like ATACs. Citation Format: Stephanie Voss, Aniko Palfi, Christoph Mueller, Andreas Pahl, Torsten Hechler, Michael Kulke. Preclinical evaluation of anti-CD37 antibody-targeted amanitin conjugates (ATAC) in B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 915.

Role of cytogenetic abnormalities detected by fluorescence in situ hybridization as a prognostic marker: Pathogenesis & clinical course in patients with B-chronic lymphocytic leukaemia.

Background & objectives:B-cell chronic lymphocytic leukaemia (B-CLL) is one of the most common forms of adult leukaemia, with a highly variable clinical course. Specific chromosomal and genetic aberrations are used clinically to predict prognosis, independent from conventional clinical markers. Molecular cytogenetic methods such as fluorescence in situ hybridization (FISH) detect aberrations in up to 80 per cent B-CLL patients. This study was conducted to score the frequencies of recurrent aberrations, i.e., del(13q14), trisomy 12, del(11q22), del(17p13), del(6q21) and IgH (immunoglobulin heavy chain) translocations and to understand their role in prognostication and risk stratification.Methods:FISH studies were performed on bone marrow aspirate or peripheral blood of 280 patients using commercially available disease-specific probe set. The data were correlated with clinical and haematological parameters such as low haemoglobin, splenomegaly and lymphadenopathy.Results:Chromosomal aberrations were detected in 79 per cent of patients, with del(13q14) (57%) as the most common cytogenetic aberration, followed by trisomy 12 (27%), del(11q22) (22%), t(14q32) (19%), del(17p13) (18%) and del(6q21) (9%). Single or in coexistence with other aberration del(13q14) had a favourable outcome in comparison to del(11q22), t(14q32), del(17p13) and del(6q21) which were associated with advanced stages of the disease. Trisomy 12 had a variable clinical course.Interpretation & conclusions:FISH was found to be a sensitive and efficient technique in detecting the prevalence of recurrent cytogenetic abnormalities. Each of these aberrations is an important independent predictor of disease progression and survival which aids in designing risk-adapted treatment strategies for better disease management.