B-cell Chronic Lymphocytic Leukemia Research Articles

Identification of Novel Therapeutic Targets in Atm-Deficient Lymphomas Using a Whole Genome CRISPR/CAS-9 Screen

ATM is a principal DNA damage response protein that synchronises a complex network of cellular responses to double stranded DNA breaks. ATM gene is recurrently mutated in a wide range of lymphoid malignancies, including B-cell chronic lymphocytic leukemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and diffuse B cell lymphoma (DLBCL). ATM pathway is utilized by many DNA damaging agents and consequently inactivation of this pathway can lead to chemoresistance. Furthermore, in the absence of ATM tumour cells exhibit genomic instability that can lead to clonal selection and evolution even under current targeted treatments. Consequently there is clear need to understand dependency pathways in ATM-deficient tumours and apply tailored targeted therapies that will specifically eliminate those tumour cells. We have previously presented a novel murine model of ATM-deficiency that spontaneously generate lymphoid tumours, mostly DLBCL. These tumours have been successfully propagated both in recipient mice and in vitro, where several cell lines have been generated. Genome editing methods, such as CRISPR/CAS-9, permit the targeted disruption of specific genes. Protocols for genome wide screens have been developed based on this technology which can be used to identify genes that are essential for cellular survival. As such, these screens can be used to identify dependency pathways for tumours with specific genetic lesions. Using lentiviral transduction we established two cell lines that stably expressed CAS-9. We then performed a genome wide CRISPR screen using the GeCKO library to identify novel therapeutic targets in these Atm-deficient tumours. This library consists of 130,209 unique single guide RNA (sgRNAs), targetting 20,611 genes including 1176 miRNAs. A comparative analysis was performed of sgRNA drop-out following 15 cellular doublings. This revealed a number of pathways including those already known to be synthetically lethal with ATM deficiency, such as ATR and PARP. Pathway analysis of the top genes from this drop-out analysis identified oxidative phosphorylation, the spliceosome, ribosome biogenesis, N-glycan biosynthesis, pyrimidine metabolism and purine metabolism as the most significantly affected pathways. Furthermore, the drop-out screen revealed a number of miRNAs, including MiR-3470a, Mir-3971, MiR-669f and MiR-719. These data provide a unique molecular assessment of the dependency of ATM-deficient lymphomas and provide a number of novel putative therapeutic targets for treating such tumours. Disclosures No relevant conflicts of interest to declare.

Ibrutinib Treatment in CLL Patients Improves T Cell Function and Blinatumomab Redirected Cytotoxicity

Introduction:T cell engaging bispecific antibodies (T-BsAb) redirect cytotoxic T cells to tumor associated antigen (TAA)-positive target cells, leading to T-cell activation and lysis of target cells. Blinatumomab is the only T-BsAb approved by FDA. Numerous T-BsAb are undergoing clinical trials, targeting both hematological malignancies (e.g. BCMA, CD123 and CD33) and solid tumors (e.g. CEA, EpCAM, and GPC3). While these T-BsAbs represent a promising novel therapeutic approach, their efficacy remains modest. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and induces durable remissions in chronic lymphocytic leukemia (CLL). Our group has reported that ibrutinib has favorable immunomodulatory effects due to inhibition of interleukin-2 inducible T-cell kinase (ITK). Ibrutinib decreased key immunosuppressive checkpoint molecules and the frequency of regulatory T cells (Treg)(Long, Beckwith et al. 2017). Moreover, Ibrutinib significantly increased the number of the activated T cells by rescuing them from activation induced cell death (AICD). However, the function of ibrutinib 'rescued' T cells and their ability to mediate T-BsAb redirected cytotoxicity is unknown. Therefore we proceeded to study how ibrutinib treatment affects the functional competency of T cells by measuring their capability to mediate Blinatumomab redirected cytotoxicity. To examine T cell function post ibrutinib treatment, T cells were isolated from pre and post treatment PBMC samples by fluorescence activated cell sorting (FACS), and used as effector cells with pre-treatment CLL B-cells as target cells. Blinatumomab redirected T-cell cytotoxicity against tumor cells and T cell survival were assayed by flow cytometry based methods. Effector cells (T cells) were labeled with CFSE. Target (CLL) cells were labeled with CellTrace Violet. They were co-cultured at an E:T ratio of 4:1 with Blinatumomab for 24 hours. Effector cells and target cells can be distinguished by different fluorescence labels. Apoptosis and cell death were evaluated by Annexin-V and PI staining. The number of viable target cells and effector cells were calculated by Countbright counting bead. Results: We observed that post-ibrutinib T cells demonstrated significantly superior cytotoxicity against autologous CLL cells in the presence of blinatumomab in three independent patient samples. Numbers of viable CLL cells co-cultured with post-ibrutinib T cells were lower when compared to those co-cultured with pre-ibrutinib T cells (33.6%, 68% and 55.1% reduction in viable CLL cells for the three patient samples, respectively). We also found that Treg depletion (by depleting CD25high/CD127low CD4+ T cells) further enhanced cytotoxicity for both pre and post-ibrutinib treatment T cells, with the numbers of viable CLL cells reduced another 30-40%. Moreover, compared to pre-ibrutinib T cells, post-ibrutinib treatment T cells again demonstrated superior cytotoxicity when Treg cells were depleted, with decreased numbers of viable CLL cells at the end of co-culture (43%, 69% and 49.4% reduction, respectively). We also observed that the numbers of viable T cells from pre-ibrutinib samples were much lower than post-ibrutinib samples (52.5%, 77.9% and 74% reduction in viable T cells, respectively). Conclusions: Our findings suggest that ibrutinib-rescued T cells are functionally competent. CLL patients' T cells post ibrutinib treatment demonstrated superior cytotoxicity against autologous leukemia cells compared to T cells from treatment baseline. Moreover, we show that increased T cell activity is not solely due to ibrutinib-related Treg depletion. Treg depletion in our experiments further enhanced the cytotoxicity of both post and pre-ibrutinib treatment T cells while post-ibrutinib T cells still demonstrated superior cytotoxicity (Fig 1). Lastly, T cells isolated from post-ibrutinib samples demonstrated significantly improved viability after in-vitro stimulation with blinatumomab, which is likely a result of reduced AICD. Disclosures Bhat: Pharmacyclics: Consultancy; Janssen: Consultancy. Rogers:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Muthusamy:Ohio State University: Patents & Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau.

Disseminated cytomegalovirus disease after bendamustine: a case report and analysis of circulating B- and T-cell subsets

BackgroundBendamustine, used for the treatment of indolent B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia, is known to cause prolonged myelosuppression and lymphocytopenia and has been associated with the risk of developing serious and fatal infections. While reports of localized CMV infections in asymptomatic patients exist, disseminated CMV disease has not been described.Case presentationWe report the first case of disseminated CMV infection in a 75-year-old male diagnosed with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia with massive bone marrow infiltration. Despite 6-cycle R-bendamustine chemotherapy resulted in a good partial response, the patient developed persistent fever and severe weight loss. Analysis of cerebrospinal fluid and peripheral blood revealed the presence of CMV-DNA, while the fundus oculi examination revealed bilateral CMV retinitis. Treatment with induction and maintenance drugs was complicated by neutropenia and deterioration of renal function with electrolyte imbalance. From an immunological standpoint, we observed a profound imbalances in phenotype and function of B- and T-cell subsets, with a high proportion of circulating total, activated CD69+ and CD80+ B-cells, a low γ/δ T-cell frequency with a high proportion of CD69- and CD38-expressing cells, and hyperactivated/exhausted CD4+ and CD8+ T-cell phenotypes unable to face CMV challenge.ConclusionsWe hereby describe a severe form of disseminated CMV disease after R-bendamustine treatment. Our observations strongly support the careful clinical monitoring of CMV reactivation/infection in oncologic patients undergoing this therapeutic regimen.

The Mithralog EC-7072 Induces Chronic Lymphocytic Leukemia Cell Death by Targeting Tonic B-Cell Receptor Signaling.

B-cell receptor (BCR)-dependent signaling is central for leukemia B-cell homeostasis, as underscored by the promising clinical results obtained in patients with chronic lymphocytic leukemia (CLL) treated with novel agents targeting components of this pathway. Herein, we demonstrate that the mithralog EC-7072 displays high ex vivo cytotoxic activity against leukemia cells from CLL patients independently from high-risk prognostic markers and IGHV mutational status. EC-7072 was significantly less toxic against T cells and NK cells and did not alter the production of the immune effector molecules IFN-γ and perforin. EC-7072 directly triggered caspase-3-dependent CLL cell apoptosis, which was not abrogated by microenvironment-derived factors that sustain leukemia cell survival. RNA-sequencing analyses revealed a dramatic EC-7072-driven reprograming of the transcriptome of CLL cells, including a wide downregulation of multiple components and targets of the BCR signaling pathway. Accordingly, we found decreased levels of phosphorylated signaling nodes downstream of the BCR. Crosslinking-mediated BCR activation antagonized CLL cell death triggered by EC-7072, increased the phosphorylation levels of the abovementioned signaling nodes and upregulated BCL2 expression, suggesting that the mithralog disrupts CLL cell viability by targeting the BCR signaling axis at multiple levels. EC-7072 exerted similar or higher antileukemic activity than that of several available CLL therapies and displayed additive or synergistic interaction with these drugs in killing CLL cells. Overall, our findings provide rationale for future investigation to test whether EC-7072 may be a potential therapeutic option for patients with CLL and other B-cell malignancies.

Immunogenicity and safety of the adjuvanted recombinant zoster vaccine in adults with haematological malignancies: a phase 3, randomised, clinical trial and post-hoc efficacy analysis

The adjuvanted recombinant zoster vaccine (Shingrix) can prevent herpes zoster in older adults and autologous haemopoietic stem cell transplant recipients. We evaluated the safety and immunogenicity of this vaccine in adults with haematological malignancies receiving immunosuppressive cancer treatments. In this phase 3, randomised, observer-blind, placebo-controlled study, done at 77 centres worldwide, we randomly assigned (1:1) patients with haematological malignancies aged 18 years and older to receive two doses of the adjuvanted recombinant zoster vaccine or placebo 1-2 months apart during or after immunosuppressive cancer treatments, and stratified participants according to their underlying diseases. The co-primary objectives of the study were the evaluation of safety and reactogenicity of the adjuvanted recombinant zoster vaccine compared with placebo from the first vaccination up to 30 days after last vaccination in all participants; evaluation of the proportion of participants with a vaccine response in terms of anti-glycoprotein E humoral immune response to the adjuvanted recombinant zoster vaccine at month 2 in all participants, excluding those with non-Hodgkin B-cell lymphoma and chronic lymphocytic leukaemia; and evaluation of the anti-glycoprotein E humoral immune responses to the vaccine compared with placebo at month 2 in all participants, excluding those with non-Hodgkin B-cell lymphoma and chronic lymphocytic leukaemia. We assessed immunogenicity in the per-protocol cohort for immunogenicity and safety in the total vaccinated cohort. The study is registered with ClinicalTrials.gov, number NCT01767467, and with the EU Clinical Trials Register, number 2012-003438-18. Between March 1, 2013, and Sept 10, 2015, we randomly assigned 286 participants to adjuvanted recombinant zoster vaccine and 283 to placebo. 283 in the vaccine group and 279 in the placebo group were vaccinated. At month 2, 119 (80·4%, 95% CI 73·1-86·5) of 148 participants had a humoral vaccine response to adjuvanted recombinant zoster vaccine, compared with one (0·8%, 0·0-4·2) of 130 participants in the placebo group, and the adjusted geometric mean anti-glycoprotein E antibody concentration was 23 132·9 mIU/mL (95% CI 16 642·8-32 153·9) in the vaccine group and 777·6 mIU/mL (702·8-860·3) in the placebo group (adjusted geometric mean ratio 29·75, 21·09-41·96; p<0·0001) in all patients, excluding those with non-Hodgkin B-cell lymphoma and chronic lymphocytic leukaemia. Humoral and cell-mediated immune responses persisted above baseline until month 13 in all strata and, as expected, vaccine was more reactogenic than placebo (within 7 days after vaccination pain was reported by 221 [79·5%] of 278 vaccine group participants and 45 [16·4%] of 274 placebo group participants; fatigue was reported by 162 [58·3%] of 278 vaccine group participants and 102 [37·2%] of 274 placebo group participants). Incidences of unsolicited or serious adverse events, potential immune-mediated diseases, disease-related events, and fatal serious adverse events were similar between the groups. The immunocompromised adult population with haematological malignancies is at high risk for herpes zoster. The adjuvanted recombinant zoster vaccine, which is currently licensed in certain countries for adults aged 50 years and older, is likely to benefit this population. GlaxoSmithKline Biologicals SA.

Association of elevated serumfree light chains with chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis

Chronic lymphocytic leukemia (CLL) and its precursor, monoclonal B-cell lymphocytosis (MBL), are heritable. Serumfree light-chain (sFLC) measures are a prognostic factor for CLL, but their role in susceptibility to CLL is not clear. We investigated differences between sFLC measurements in pre-treatment serum from five groups to inform the association of sFLC with familial and sporadic CLL: (1) familial CLL (n = 154), (2) sporadic CLL (n = 302), (3) familial MBL (n = 87), (4) unaffected first-degree relatives from CLL/MBL families (n = 263), and (5) reference population (n = 15,396). The percent of individuals having elevated monoclonal and polyclonal sFLCs was compared using age-stratified and age- and sex-adjusted logistic regression models. In age groups >50 years, monoclonal sFLC elevations were increased in sporadic and familial CLL cases compared to the reference population (p’s < 0.05). However, there were no statistically significant differences in sFLC monoclonal or polyclonal elevations between familial and sporadic CLL cases (p’s > 0.05). Unaffected relatives and MBL cases from CLL/MBL families, ages >60 years, showed elevated monoclonal sFLC, compared to the reference population (p’s < 0.05). This is the first study to demonstrate monoclonal sFLC elevations in CLL cases compared to controls. Monoclonal sFLC levels may provide additional risk information in relatives of CLL probands.

TRIM13 inhibited cell proliferation and induced cell apoptosis by regulating NF-κB pathway in non-small-cell lung carcinoma cells

Tripartite Motif Containing 13 (TRIM13), a member of TRIM proteins, is deleted in multiple tumor types, especially in B-cell chronic lymphocytic leukemia and multiple myeloma. The present study explored the expression and potential role of TRIM13 in non-small-cell lung carcinoma (NSCLC). We found that TRIM13 mRNA and protein expression was reduced in NSCLC tissues and cell lines in comparison to paired non-cancerous tissues and a human normal bronchial epithelial cell line, respectively. Overexpression of TRIM13 in NCI-H1975 and SPC-A-1 cells hampered cell proliferation. Additionally, TRIM13 overexpression increased the levels of cleaved caspase-3. TRIM13-induced NSCLC cell apoptosis was attenuated by a caspase-3 inhibitor Ac-DEVD-CHO, suggesting that TRIM13 induced cell apoptosis partially through a caspase-3-dependent pathway. Moreover, it has been reported that TRIM13 can regulate nuclear factor kappaB (NF-κB) activity. Our data showed that TRIM13 overexpression inactivated NF-κB as indicated by the increased cytosolic NF-κB and decreased nuclear NF-κB. Exposure to an NF-κB inhibitor PDTC significantly blocked the impact of TRIM13 knockdown on cell proliferation and apoptosis, indicating the functions of TRIM13 in NSCLC cells were mediated by the NF-κB pathway. Finally, we demonstrated that TRIM13 overexpression suppressed tumor growth and induced cell apoptosis in vivo by using a xenograft mouse model. Collectively, our results indicate that TRIM13 behaves as a tumor suppressor in NSCLC through regulating NF-κB pathway. Our findings may offer a promising therapeutic target for NSCLC.

Obinutuzumab and ibrutinib induction therapy followed by a minimal residual disease-driven strategy in patients with chronic lymphocytic leukaemia (ICLL07 FILO): a single-arm, multicentre, phase 2 trial

In patients with chronic lymphocytic leukaemia, achievement of a complete response with minimal residual disease of less than 0·01% (ie, <1 chronic lymphocytic leukaemia cell per 10 000 leukocytes) in bone marrow has been associated with improved progression-free survival. We aimed to explore the activity of induction therapy for 9 months with obinutuzumab and ibrutinib, followed up with a minimal residual disease-driven therapeutic strategy for 6 additional months, in previously untreated patients. We did a single-arm, phase 2 trial in 27 university hospitals, general hospitals, and specialist cancer centres in France. Eligible patients were at least 18 years old and previously untreated, and had immunophenotypically confirmed B-cell chronic lymphocytic leukaemia; an Eastern Cooperative Oncology Group (ECOG) performance status score of less than 2; a Binet stage C according to IWCLL 2008 criteria or Binet stage A and B with active disease; no 17p deletion or absence of p53 mutation; and were considered medically fit. In the first part of the study (induction phase), all participants received eight intravenous infusions of obinutuzumab 1000 mg over six 4-weekly cycles and oral ibrutinib 420 mg once per day for 9 months. In part 2, after assessment on day 1 of month 9, patients with a complete response and bone marrow minimal residual disease of less than 0·01% received only oral ibrutinib 420 mg once per day for 6 additional months. Patients with a partial response, or with a complete response and bone marrow minimal residual disease of 0·01% or more, received 6 months of four 4-weekly cycles of intravenous fludarabine, cyclophosphamide, and obinutuzumab 1000 mg, alongside continuing ibrutinib 420 mg once per day. The primary endpoint was the proportion of patients achieving a complete response with bone marrow minimal residual disease less than 0·01% on day 1 of month 16 assessed by intention to treat (ITT). This trial is registered with ClinicalTrials.gov (number NCT02666898) and is still open for follow-up. Between Oct 27, 2015, and May 16, 2017, 135 patients were enrolled. After induction treatment (day 1 of month 9), 130 patients were evaluable, of which ten (8%) achieved a complete response with bone marrow minimal residual disease of less than 0·01% and were assigned to ibrutinib, and 120 (92%) were assigned to ibrutinib plus fludarabine, cyclophosphamide, and obinutuzumab. After minimal residual disease-guided treatment (day 1 of month 16), 84 (62%, 90% CI 55-69) of 135 patients (ITT population) achieved a complete response with bone marrow minimal residual disease of less than 0·01%. The most common haematological adverse event was thrombocytopenia (in 45 [34%] of 133 patients at grade 1-2 in months 1-9 and in 43 [33%] of 130 patients at grade 1-2 in months 9-15). The most common non-haematological adverse events were infusion-related reactions (in 83 [62%] patients at grade 1-2 in months 1-9) and gastrointestinal disorders (in 62 [48%] patients at grades 1 and 2 in months 9-15). 49 serious adverse events occurred, most frequently infections (ten), cardiac events (eight), and haematological events (eight). No treatment-related deaths occurred. Obinutuzumab and ibrutinib induction therapy followed by a minimal residual disease driven strategy is safe and active in patients with previously untreated chronic lymphocytic leukaemia. With longer follow-up, including assessing the evolution of minimal residual disease, if response is maintained, this strategy could be an option in the first-line setting in patients with chronic lymphocytic leukaemia, although randomised evidence is needed. Roche, Janssen.

Abstract 2318: Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition

Abstract The first Chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T cells eliminate all B-lineage cells, including non-malignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections.Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T cells and subsequent testing of the CAR T cells in various in vitro assay with target cells, demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in target cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T cells, and our tests confirmed this. To improve IGK CAR T cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs. Hence, enabling T cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity. Citation Format: Hakan Köksal, Pierre Dillard, Sólrún M. Maggadóttir, Gunnar Kvalheim, Erlend B. Smeland, June H. Myklebust, Else Marit Inderberg, Sébastien Walchli. Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2318.

Focus on pre-processing step to ensure the clinical transferability of Raman data acquired on lymphocytes in different experimental and instrumental conditions

The efficiency of Raman spectroscopy for the analysis of biomedical samples has been largely demonstrated at the proof-of-concept stage during one-off measurement campaigns. However, bringing these results to the patient bedside requires to fill the gap for transferability of Raman data acquired in different experimental and instrumental conditions during multicentric measurement campaigns. In this study, we propose to evaluate a solution consisting in the application of Raman data preprocessing specifically developed to remove the spectral variability induced by such different conditions. For this purpose, we compared Raman data of lymphocytes acquired during two independent measurement campaigns from fresh unstained glass blood smears originating from healthy individuals and patients with a B-cell chronic lymphocytic leukemia (CLL) at an advanced stage. The differences between these campaigns were i) the instrumental configuration of the Raman devices, ii) the hospital partner, iii) the smear preparation method. A preprocessing developed previously and efficient for a specific measurement campaign is shown obsolete for these multicentric data. A second preprocessing based on Extended Multiplicative Signal Correction is able to homogenize the data by neutralizing the signal variability between the two measurement campaigns. These conclusions are drawn from the analysis of the data by Principal Component Analysis to study the source of variability between the two campaigns and by Partial Least Squares – Discriminant Analysis to assess the separability between healthy and CLL patients.

PB1915 CD14 AS A POTENTIAL PROGNOSTIC FACTOR AND BCL‐2 AS A THERAPEUTIC TARGET IN EGYPTIAN B‐CELL CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

Please indicate where the abstract has been published before: Journal of Applied Hematology.Background:B‐cell chronic lymphocytic leukemia (B‐CLL) is a unique lymphoproliferative disorder that scarcely occurs under the age of 40. B‐CLL represents a neoplastic disorder caused primarily by defective programmed cell death and accompanied by a myriad of cellular and humoral immune defects.Aims:This study aimed to assess Bcl‐2 and CD14 expression in B‐CLL patients and their study as probable prognostic and therapy targeting factors.Methods:In this study, we assessed Bcl‐2 and cluster of differentiation (CD14) expression in a group of Egyptian patients with B‐CLL. Forty B‐CLL patients and 20 apparently healthy individuals served as the control group were included in this study.Results:Aberrant expression of Bcl‐2 protein appeared in all B‐CLL patients (100%). Bcl‐2 expression showed a highly positive correlation with total lymphocyte count and lymphocyte count (P = .000 for both) and a positive correlation with lactate dehydrogenase (P = 0.044). The expression of myelomonocytic antigen “CD14” above the cutoff value 5 × 109/L was reported in 70% (28/40) of B‐CLL patients, 55.6% (10/18) of the intermediate‐risk group, and 81.8% (18/22) of high‐risk group.Summary/Conclusion:There was a significant increase in Bcl‐2 protein and CD14 in B‐CLL patients. Bcl‐2 was highly increased in all patients and CD14 more observed in the high‐risk group than that of the intermediate risk group.

Epidemiology, clinical and cytological features of lymphoma in Boxer dogs.

The aim of this study was to evaluate the epidemiology, clinical and laboratory characteristics of canine lymphomas as well as some aspects of treatment outcomes. The study was conducted on Boxer dogs with lymphoma diagnosed by cytology and immunocytochemistry (CD3 and CD79 alpha). During the study period, lymphoma was diagnosed in 63 Boxers; 86.8% were T-cell (based on the Kiel classification: small clear cell lymphoma, pleomorphic small cell lymphoma, pleomorphic mixed T-cell lymphoma, pleomorphic large T-cell lymphoma, lymphoblastic lymphoma/acute lymphoblastic leukaemia) and 13.2% were B-cell lymphomas (according to the Kiel classification: B-cell chronic lymphocytic leukaemia, centroblastic/centroblastic polymorphic lymphoma). Overall survival (OS) was significantly longer in dogs with low-grade than with high-grade lymphoma (median OS of 6.8 and 4.7 months, respectively; P = 0.024). OS was not influenced by WHO clinical stage, WHO clinical substage, presence of splenomegaly, early administration of glucocorticoids or the time from the first presentation to the beginning of chemotherapy. There are no significant differences in clinical and laboratory parameters between low-grade and high-grade lymphomas. Boxer dogs are predisposed to T-cell lymphoma, with a predominance of high-grade tumour, especially pleomorphic, mixed small and large T-cell subtype. It is possible that Boxer dogs may respond less favourably to chemotherapy than patients of other breeds.

Frequency of Zap-70 and CD38 in Newly Diagnosed Cases of B-Cell Chronic Lymphocytic Leukemia

Frequency of Zap-70 and CD38 in Newly Diagnosed Cases of B-Cell Chronic Lymphocytic Leukemia

Mechanism for IL-15-Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins.

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.

Analysis of stochastic Nicholson-type delay system with patch structure

This paper is devoted an investigation of a stochastic Nicholson-type delay system with patch structure, which includes the models of marine protected areas and B-cell chronic lymphocytic leukemia dynamics all affected by some stochastic perturbation. Firstly, we prove that the system has a unique global positive solution by constructing a suitable Lyapunov functional. Then we show that the system is ultimately bounded in probability and the average in time of the second moment of solution is bounded. Furthermore, we give asymptotic pathwise estimation of the solution under some conditions. Finally, numerical simulations confirm the theoretical results. Our results improve and generalize previous related results.

IL-15-driven clonal expansion of TLR9-primed human B-CLL is characterized by activation of AKT and STAT5, downstream elevation of cyclin D2 and repression of DNA damage response mediators, ATM and TP53BP

Abstract Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) is linked to mutagenesis and occurs within lymphoid tissues. CpG DNA (ODN-2006) and IL-15, a cytokine found within stromal cells of B-CLL-infiltrated tissues, manifest striking synergy in promoting in vitro growth of B-CLL cells (J Immunol 195:901–923, 2015). Synergy is in part explained by an ODN-induced, NF-kB (p65)-dependent elevation of IL-15Rα and IL-2/15Rβ (CD122) mRNA and protein (J Immunol 201:1570–1585, 2018). Most recently, we examined the downstream consequences of IL-15 signaling through experiments involving intracellular immunofluorescence staining, pharmacologic inhibitors, RT-PCR, and western blotting. Levels of activated pAKTSer473 and pSTAT5Tyr694 were significantly elevated soon after IL-15 exposure to ODN-primed B-CLL cells; furthermore, STAT5 remained activated throughout cycling. Pharmacologic inhibitors of PI-3K or STAT5 blocked IL-15-driven growth, indicating the functional relevance of these pathways. RT-PCR experiments +/− these inhibitors show that IL-15 signaling in B-CLL is characterized by PI-3K &amp; STAT5-dependent rise in cyclin-D2 and repression of two important DNA damage response (DDR) mediators, ATM and TP53BP1, known to dampen growth and facilitate DNA repair. Repression of the latter two proteins was confirmed by western blotting and immunofluorescence studies. We conclude that IL-15 signaling in TLR9-primed B-CLL can provoke a rapid burst in growth via mechanisms resembling those recently described in CD8 T cells. Nonetheless, because dividing B-CLL cells additionally upregulate the mutagenic enzyme, AICDA (Blood 120: 4802–4811, 2012), these simultaneous events may augment B-CLL mutagenesis.

The actin‐binding protein cortactin controls transendothelial migration and organ infiltration of acute lymphoblastic leukemia T‐cells

T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive neoplasm due to high rates of organ infiltration and relapse, which are major obstacles for curing the disease. Infiltration of T‐ALL cells requires extravasation, a migratory process highly dependent on actin cytoskeleton dynamics. Cortactin is an actin‐binding protein that regulates actin remodeling and thus cell migration. High expression of cortactin is associated with invasiveness in several types of solid tumors, and recently, expression of cortactin in adult B‐cell chronic lymphocytic leukemia was correlated with negative prognostic factors. However, it is unknown whether cortactin plays a role in T‐ALL biology. Here, we analyzed cortactin expression in the murine and human T‐ALL cell lines 6645/4 and Jurkat, and cortactin functions using different in vitro and in vivo methods such as transendothelial migration, bone marrow (BM) colonization and xenotransplantation assays. Compared to normal T cells, leukemic T cells expressed significantly higher levels not only of cortactin but also of the integrin VLA‐4 and CXCR4, the receptor for the T cell chemokine CXCL12, which are both required for T cell extravasation. Leukemic T cells transmigrated significantly more, and cortactin levels were significantly higher in cells capable of transmigrating across endothelial monolayers towards CXCL12. Of note, only leukemic T cells with highest cortactin levels were capable of invading BM‐like organoids. Preliminary in vivo assays showed that leukemic T cells had different levels of cortactin depending on the infiltrated organ. Surprisingly, once leukemic T cells completed infiltration they diminished cortactin expression suggesting that cortactin is needed for initial organ invasion, but not for subsequent functions. Taken together, our results highlight an important role of cortactin in T‐ALL infiltration; and contribute to a better understanding of T‐ALL aggressiveness. Thus, cortactin may be a potential pharmacological target to prevent T‐ALL organ infiltration.Support or Funding InformationCONACyTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibiting Bruton's Tyrosine Kinase in CLL and Other B-Cell Malignancies.

Inhibitors of Bruton's tyrosine kinase (BTK), a major kinase in the B-cell receptor (BCR) signaling pathway, mediating B-cell proliferation and apoptosis, have substantially altered the management, clinical course, and outcome of patients with B-cell malignancies. This is especially true for patients with previously limited treatment options due to disease characteristics or coexisting diseases. Ibrutinib was the first orally available, nonselective and irreversible inhibitor of BTK approved for the treatment of patients with various B-cell malignancies. Newer and more selective BTK inhibitors are currently in clinical development, including acalabrutinib, which is currently US FDA approved for previously treated mantle cell lymphoma. Significant efforts are underway to investigate the optimal combinations, timing, and sequencing of BTK inhibitors with other regimens and targeted agents, and to capitalize on the immunomodulatory modes of action of BTK inhibitors to correct tumor-induced immune defects and to achieve long-lasting tumor control. This review describes the major milestones in the clinical development of BTK inhibitors in chronic lymphocytic leukemia and other B-cell malignancies, highlights the most recent long-term follow-up results, and evaluates the role of BTK inhibitors and their combination with other agents in B-cell malignancies and other indications.

Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation

AbstractWe have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab′)2 antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.

Protoporphyrin IX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and induces apoptosis in B-cell chronic lymphocytic leukemia cells

p53 is a tumor suppressor, which belongs to the p53 family of proteins. The family consists of p53, p63 and p73 proteins, which share similar structure and function. Activation of wild-type p53 or TAp73 in tumors leads to tumor regression, and small molecules restoring the p53 pathway are in clinical development. Protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid, is a clinically approved drug applied in photodynamic diagnosis and therapy. PpIX induces p53-dependent and TAp73-dependent apoptosis and inhibits TAp73/MDM2 and TAp73/MDM4 interactions. Here we demonstrate that PpIX is a dual inhibitor of p53/MDM2 and p53/MDM4 interactions and activates apoptosis in B-cell chronic lymphocytic leukemia cells without illumination and without affecting normal cells. PpIX stabilizes p53 and TAp73 proteins, induces p53-downstream apoptotic targets and provokes cancer cell death at doses non-toxic to normal cells. Our findings open up new opportunities for repurposing PpIX for treating lymphoblastic leukemia with wild-type TP53.