Lymphocytic Leukemia B-cell Research Articles

Transgenic HA-1-Specific CD8+ T-Lymphocytes Selectively Target Leukemic Cells

A significant share of allogeneic hematopoietic stem cell transplantations (allo-HSCT) results in the relapse of malignant disease. The T cell immune response to minor histocompatibility antigens (MiHAs) promotes a favorable graft-versus-leukemia response. The immunogenic MiHA HA-1 is a promising target for leukemia immunotherapy, as it is predominantly expressed in hematopoietic tissues and presented by the common HLA A*02:01 allele. Adoptive transfer of HA-1-specific modified CD8+ T cells could complement allo-HSCT from HA-1- donors to HA-1+ recipients. Using bioinformatic analysis and a reporter T cell line, we discovered 13 T cell receptors (TCRs) specific for HA-1. Their affinities were measured by the response of the TCR-transduced reporter cell lines to HA-1+ cells. The studied TCRs showed no cross-reactivity to the panel of donor peripheral mononuclear blood cells with 28 common HLA alleles. CD8+ T cells after endogenous TCR knock out and introduction of transgenic HA-1-specific TCR were able to lyse hematopoietic cells from HA-1+ patients with acute myeloid, T-, and B-cell lymphocytic leukemia (n = 15). No cytotoxic effect was observed on cells from HA-1- or HLA-A*02-negative donors (n = 10). The results support the use of HA-1 as a target for post-transplant T cell therapy.

Acute Lymphocytic Leukemia in a child presenting as a solitary intracranial epidural mass


 Acute Lymphocytic leukemia (ALL) is the most common childhood malignancy, CNS involvement occurs in 3-5% of the patients. We present a case of a 14-year-old boy who presented to us with nighttime fever, headache, and right-sided focal fits. Reports showed decreased platelets, increased WBC with predominant eosinophils and imaging showed a subgaleal, epidural and subdural collection. Bone marrow biopsy and CSF analysis were negative for any hematological malignancy. A biopsy and histopathology of the CNS lesion confirmed the diagnosis of Precursor B-cell Lymphocytic Leukemia. Although CSF analysis is useful for evaluating CNS involvement, ALL cannot be ruled out by negative CSF reports

Detection and Characterization of Paraproteinemia in Canine Chronic B-cell Lymphocytic Leukemia Using Routine and Free Light Chain Immunofixation.

Hyperglobulinemia is reported in 26% of canine chronic B-cell lymphocytic leukemia (B-CLL) cases. However, few cases have been characterized by protein electrophoresis and immunofixation (IF), and the incidence of a monoclonal protein (M-protein) is unknown using these techniques. To characterize and determine the proportion of canine B-CLL cases with an M-protein using plasma protein electrophoresis (PPE), routine and free light chain (fLC) IF, and to assess if productive B-CLL cases express MUM1/IRF4 by cell tube block (CTB). PPE, routine (targeting IgG, IgA, IgM, IgG4, and light chain) and fLC IF were performed using 48 dog B-CLL plasma samples from patients diagnosed via peripheral blood flow cytometry. CTB was performed on a separate cohort of 15 patients. Hyperproteinemia (>7.5g/dL) was present in 17/48 cases (35%). An M-protein was detected in 32/48 cases (67%). Of these, 19/32 cases (59%) had only complete (monoclonal heavy and light chain) M-proteins detected, 10/32 cases (31%) had both complete and fLC M-proteins detected, and 3/32 cases (9%) had only an fLC M-protein detected. IgM was the most common clonal immunoglobulin isotype detected (23 cases). CD21+ cell counts were higher in cases with detectable M-protein. Plasma fLC IF suggested β-γ region interference, likely caused by clotting proteins. All B-CLL cases consistently expressed PAX5 and did not express MUM1/IRF4. Most B-CLL cases had an M-protein and were not hyperproteinemic. Most cases with paraproteins had a complete IgM monoclonal gammopathy; a subset had documented fLCs. The prognostic significance of heavy and fLC presence should be evaluated.

Abstract 1033: Adult B-cell lymphocytic leukemia associated with osteolysis but normocalcemia and normal hematologic parameters - rare case or influenced by mifepristone treatment

Abstract Acute B-cell lymphoblastic leukemia (All) is much less common in adults than children and is much less common to present with bone pain. Extremely rare, but precedented, is the absence of hematologic abnormalities or lymphadenopathy. In some of the rare cases early diagnosis was achieved because hypercalcemia and bone pain were related to ectopic production of parathyroid-related peptide with consequent hypercalcemia and bone pain from hyperparathyroidism. However, there has been a case report in a 37-year-old male with B-cell lymphoblastic leukemia with osteolytic lesions, normal parathyroid hormone levels, and normal hematologic parameters despite >80% of the bone marrow replaced with blast cells. We present another case of a 55-year-old woman presenting with B cell ALL, bone pain and perfectly normal hematologic parameters despite 95% of the marrow replaced with blast cells. Her pain began 2 ½ months prior to the bone biopsy which was done because of the finding of osteolytic lesions on MRI. Her first bone marrow evaluation was performed 1 ½ months later. Related to further delays in even getting an appointment with the proper oncologist she was started on mifepristone 200mg daily to hopefully help delay a hematologic ALL crisis in view of 95% blast cells on biopsy. After 1 month of mifepristone therapy her CBC was still perfectly normal before standard chemotherapy was started. Mifepristone was started because of its effectiveness in improving length and quality of life in patients with a variety of advanced cancers without any more treatment options coupled with its beneficial effect in controlled studies with spontaneous leukemia in mice. Furthermore, the target for mifepristone, the immunomodulatory protein known as the progesterone induced blocking factor (PIBF), was found to be highly expressed by multiple human leukemia cell lines and was suppressed by mifepristone. The most common clinical state found in patients with advanced cancers following treatment with mifepristone is to stabilize the disease and inhibit further spread. The possibility exists that the failure to progress to typical pancytopenia after 95% of the bone marrow was replaced by blast cells was related to the mifepristone treatment. However, the possibility also exists that some unknown factor inhibiting this progression was operative as in some other very rare reported cases. With the possibility of a beneficial effect of treatment, mifepristone, an extremely well tolerated oral agent, could be considered for treating ALL when no other reasonable treatment options are available, e.g., a patient not likely to survive standard therapy or failure to respond to traditional chemotherapy. Even in this patient, if it takes more than 1 month to induce remission, her 40% 5-year survival will even be lower so one could consider maintenance therapy with mifepristone. Citation Format: Jerome H. Check. Adult B-cell lymphocytic leukemia associated with osteolysis but normocalcemia and normal hematologic parameters - rare case or influenced by mifepristone treatment [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 1033.

Flow cytometry detection and quantification of CAR T cells into solid tumors.

Adoptive T cell therapy (ACT) is a therapeutic approach which employs genetically manipulated autologous T cells to target and eliminate a patient's malignancy. This novel therapeutic approach, when employing a chimeric antigen receptor (CAR) targeting CD19-expressing B cells, has shown remarkable success in treating acute B-cell lymphocytic leukemia. However, blood born malignancies represent only a fraction of cancers which affect patients. Unfortunately, the utilization of ACT to target solid malignancies has only shown marginal success rates. There are many known obstacles which hinder CAR T cell therapy in patients suffering from solid cancer, one notable obstacle is the effective trafficking of CAR T cells to the tumor site. With the rapid advancement of novel approaches and targets which may enhance CAR T cell infiltration into solid tumors, a standardized approach to assess and measure CAR T cell infiltration becomes imperative in order to compare these different approaches across platforms. Here we describe a flow cytometry method which enables the rapid detection and quantification of CAR T cells which have reached and entered the tumor mass following intravenous injection. Competence with single cell preparation and flow cytometry is required for optimal results.

The role of cytogenetic and molecular testing in the diagnostics and prognostication of chronic B-cell lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) is the most frequently diagnosed leukemia in an adult population in Europe and North America. Disease pathogenesis is not well defined. The majority patients are following the “wait and watch” strategy since early treatment does not affect survival. The therapeutic decision is based on the clinical stage of disease, presence of comorbid conditions clinical disease activity as well as 11q deletion and 17p deletion status and/or mutation in the TP53 gene. Moreover, expression of CD38, ZAP70, and mutational status of IGVH gene are well-known prognostic factors. The following chromosomal abnormalities are the most frequently diagnosed in CLL: 13q14 (in 50 – 60% CLL), 11q22-23 (in 12 – 18% CLL), 17p13 (in 10% CLL) deletion, 6q (in about 6% CLL) and trisomy of chromosome 12 (in 10 – 20% CLL). However, the above-mentioned factors are not able to define all, high-risk patients. Therefore, there is an urgent need to search for new prognostic and predictive factors, which might be helpful in better classification and selection for personalized therapy for B-cell CLL patients. This prompted us to review both the well-known and new prognostic/predictive factors.

KPMW135, a Biosuperior CD3 Bispecific Version of Rituximab Created By a Novel Chemical Conjugation Technology Demonstrates Increased Anti-Tumor Activity By Adding T Cell-Mediated Cytotoxicity Activity to the Existing Mechanisms of Rituximab

Background We describe KPMW135, a novel CD20 x CD3 bispecific molecule, in which a CD3-directed ScFv (SP34) is conjugated to rituximab (RTX). RTX is a chimeric monoclonal antibody (mAb) specific for CD20, used to treat B-cell lymphomas and lymphocytic leukemias. Monoclonal Ab therapy enhancer (MATE™) enables efficient site-directed chemical conjugation to "off-the-shelf" therapeutic mAbs and allows the development of bispecific therapeutic candidates. Our MATE platform allows for the chemical engineering of existing Abs without the need to create new DNA vectors or genetic engineering of master cell lines. This approach is superior to previous conjugation methods that 1) lack site-directed conjugation specificity by indiscriminately binding to available amino acid residues or 2) require genetic engineering to create conjugate tags. Methods SP34 scFv was constructed using published CDRs. The expression was in FreeStyle 293 cells, followed by affinity purification and SEC to obtain monomeric scFv. The binding of CD20 to KPMW135 was measured by Bio-Layer Interferometry using Octet (Fortebio). Equilibrium dissociation constant (Kd) was calculated using the Octet HT software. The binding of CD3εδ, CD16a, and FcRn were determined by ELISA using neutravidin-coated plates. Biotinylated Human CD3ε & CD3δ heterodimer protein (Avi tag), CD16a (Avi Tag) and human CD16a were used. Readout was determined with anti-human F(ab) HRP. For in vitro T cell-mediated cytotoxicity assays, unfractionated and NK-cell depleted PBMCs were prepared from freshly-thawed and PHA + IL-2 prestimulated PBMCs. Daudi (CD20+) B lymphoblast cells were engineered to stably express a beta-gal reporter fragment using KILR retroparticles (Eurofins). Target cells were treated with KPMW135, RTX, and controls. PBMCs were introduced at an effector:target ratio of 15:1 and incubated for 18h. Luminescence signal was obtained with luminometer to reflect target cell death. Cynomolgus monkeys were intravenously injected with RTX or KPMW135 (30 μg/kg). Endpoints included clinical observations, cytokine profile and flow cytometric immunophenotyping of T cells, monocytes, granulocytes, NK cells, and B cells (CD45, CD3, CD16, CD14, NKG2A, HLA DR) and cellular activation (CD44 and CD69). Results Binding affinity of CD20 to KPMW135 was 0.75nM. CD20 binding to RTX and KPMW135 was similar, thus conjugation of the CD3-binding ScFv did not negatively affect affinity to CD20. ELISA results show KPMW135 binds to CD3εδ, FcRn, and CD16a, indicating that the conjugation does not interfere with binding to their various moieties. In vitro T cell-mediated cytotoxicity results show the activity of Fc/FcR and CD20 binding by the RTX moiety versus CD3 binding to effector T cells. T cell enrichment by functionally prestimulated PBMCs increased killing of target cells by 2-fold. Mechanically depleted NK cells verified that target cell death was induced by T cells, rather than by NK cells via ADCC. KPMW135 elicited target cell killing with an EC50 of 0.03-0.07nM or 0.09-021nM for prestimulated or freshly thawed PBMCs, respectively. Studies in Cynomolgus monkeys showed that KPMW135 can activate T cells in vivo as shown by increased CD69 and CD44 expression (3- and 2-fold, respectively) with a peak at 4h post-dose compared to equivalent dose RTX. A pronounced depletion of B cells was observed in KPMW135 treated animals as early as half hour post-dose, with a partial recovery by days 7-14, while an equivalent dose of RTX induced a transient B-cell depletion followed by a quicker return to baseline levels. Conclusions KPMW135 is a CD20 x CD3 bispecific created by chemical conjugation of CD3-specific ScFv to "off-the-shelf" RTX using our proprietary MATE™ technology, which maintains RTX native binding to FcRs via the Fc domain and its native binding to CD20 on target cells. KPMW135 retains RTX native ability to target B lymphoid malignancies via Fc effector mechanisms like ADCC and ADCP, but now adds T cell-mediated cytotoxicity. Our in vitro data shows increased target cell killing by KPMW135 compared to RTX, and our in vivo study confirms KPMW135 ability to induce target dependent T cell activation and recruitment, making it superior to unconjugated RTX. CD3 MATEs generated with other Abs show similar increased T cell-mediated cytotoxicity, confirming that this technology can be applied to generate "off-the-shelf" biosuperior antibodies. Disclosures Vidal: Kleo Pharmaceuticals: Current Employment, Current equity holder in private company. Cukan:Kleo Pharmaceuticals: Current Employment. Vaill:Kleo Pharmaceuticals: Current Employment. Bunin:Kleo Pharmaceuticals: Current Employment. Rossi:Kleo Pharmaceuticals: Current Employment. Iben:Kleo Pharmaceuticals: Current Employment. Trinh:Kleo Pharmaceuticals: Current Employment. McGrath:Kleo Pharmaceuticals: Current Employment. Berbasova:Kleo Pharmaceuticals: Current Employment. Alvarez:Kleo Pharmaceuticals: Current Employment. Rastelli:Kleo Pharmaceuticals: Current Employment. Welsch:Kleo Pharmaceuticals: Ended employment in the past 24 months.

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2’s hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366’s cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

Abstract 6466: Serum immunoglobulins are an independent prognostic marker of time to first therapy in newly diagnosed chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL)

Abstract Immune dysfunction is a hallmark of CLL/MBL; ~25% individuals with newly diagnosed CLL have hypogammaglobulinemia at diagnosis. In this study of newly diagnosed CLL/MBL, we sought to a) investigate the association between serum immunoglobulins and specific mutations identified by next generation sequencing, and b) determine if serum immunoglobulin levels predict TFT, independent of the CLL-International Prognostic Index (CLL-IPI) and tumor mutational load (TML). We used the Mayo Clinic CLL Resource to identify newly diagnosed CLL/MBL individuals consented within 2 years of diagnosis. Baseline clinical characteristics and the CLL-IPI score was ascertained on all individuals. DNA was extracted from peripheral blood CD5+/CD19+ clonal B-cells; and the entire coding regions of 60 somatically recurring mutated CLL driver genes were enriched using a customized Agilent SureSelect gene panel and sequenced in Illumina Hiseq4000. Serum immunoglobulins were quantitated by radial immunodiffusion using Immunoplates. The relationship between serum immunoglobulins was assessed: a) with individual gene mutations (mutated vs. not); and b) using the TML score (using a value of 0, 1, and 2 or greater [2+] mutated genes). The impact of serum immunoglobulin levels on TFT was analyzed for all individuals. Of 356 individuals identified, 80 (22%) had MBL, median age was 63 years (28-87), 261 (73%) were male, and the CLL-IPI distribution was: low risk (n=149, 42%), intermediate risk (n=112, 32%), and high/very high risk (n=94, 26%). We observed 43%, 39%, and 18% individuals with 0, 1, or 2+ mutated genes, respectively. The most commonly mutated genes were NOTCH1 (13%), SF3B1 (13%), TP53 (8%), ATM (8%), BIRC3 (7%), and NFKBIE (7%). Using standard cutoffs, 135 (38%) individuals had low serum IgG, 73 (21%) had low serum IgA, and 154 (44%) had low serum IgM at diagnosis. There were no differences in individual gene mutation frequencies between individuals with low and normal serum IgG. In contrast, individuals with low serum IgA were more likely to have mutations in NOTCH1 (25% vs 10%) and ATM (14% vs 7%) compared to those with normal IgA, and individuals with low serum IgM were more likely to have mutations in NOTCH1 (19% vs 9%) and BIRC3 (11% vs 4%) compared to those with normal IgM (all p-values <0.05). There were no significant differences in individuals with low serum IgG based on TML scores of 0, 1, and 2+ (31%, 35%, 40%, respectively, p=0.28). However, individuals with low serum IgA and low serum IgM were more likely to have a higher TML (8%, 20%, 36% for IgA, p<0.0001; and 35%, 45%, 54% for IgM, p<0.0001, for TML scores of 0, 1, and 2+, respectively). After a median follow-up of 6.9 years, 157 individuals required CLL therapy. After adjusting for CLL-IPI and TML, low serum IgG, low serum IgA, and low serum IgM (hazard ratio [HR]: 1.8, 2.3, and 2.4, respectively, all p-values <0.05) were independently associated with a shorter TFT. Our study shows that individuals with CLL/MBL who harbor mutations in NOTCH1, ATM and BIRC3 are more likely to have low serum IgA and IgM at diagnosis. Serum IgA and IgM (but not serum IgG) levels decrease with an increase in the TML score. Finally, low serum immunoglobulins can predict TFT, independent of the CLL-IPI and TML, which has important implications for risk stratification in newly diagnosed CLL/MBL. Citation Format: Sameer A. Parikh, Cristine Allmer, Geffen Kleinstern, Nicholas J. Boddicker, Shulan Tian, Cecilia B. De Campos, Laura A. Bruins, Daniel R. o"brien, Brian F. Kabat, Kari G. Rabe, Aaron D. Norman, Huihuang Yan, Xing Li, Timothy G. Call, Wei Ding, Jose F. Leis, Saad J. Kenderian, Celine M. Vachon, James R. Cerhan, Neil E. Kay, Susan L. Slager, Esteban Braggio. Serum immunoglobulins are an independent prognostic marker of time to first therapy in newly diagnosed chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6466.

Antiproliferative Effect of Green Papaya on Lymphocytic Leukemic Cells

Numerous edible plants have been reported to interfere with the carcinogenic process, and therefore, the regular consumption of these plant products may reduce the risk of developing cancer. We investigated the effect of papaya fruit and leaves on the cell proliferation of Jurkat T-lymphocytic and Daudi B-lymphocytic leukemia cells. Cells were treated with aqueous or methanolic extracts from leaves, skin, pulp, and seeds from green papaya. The papaya fractions were tested for total phenolic content, total flavonoids content, and anti-oxidation activity using chemical assays. Cell proliferation was measured using a WST-1 assay. Our data indicate that methanol and water extracts of seeds and leaves contained higher concentrations of total phenolic compounds and higher anti-oxidation activity than that of extracts from skin and pulp. Both methanol and water extracts from leaves and skin potently inhibited the proliferation of leukemic Jurkat T-cells and Daudi B-cells. However, the effect was more potent on Jurkat T-cells, and the leaf extracts were more effective than that of skin extracts. None of the pulp or seed extracts showed inhibitory activity on leukemic cell proliferation. Although papaya leaves are not consumed as a food, leaf extracts have been used for the treatment of various conditions, including dengue and malaria fevers, gastric ulcers, low platelet counts, and cancers of the breast, lung, and cervix. Our data suggest that the consumption of papaya leaf extracts may also be beneficial in preventing and/or treating lymphocytic cancer. Isolation of active compounds from papaya leaves will also help in developing new drugs for cancer treatment.

Clinical Value of NOTCH1 Mutations Detection among Chronic Lymphocytic Leukemia Patients.

Background:The data about the clinical impact of NOTCH1 mutations among Egyptians B – cell chronic lymphocytic patients is not previously identified. We herein, evaluate the prevalence and the prognostic significance of neurogenic locus notch homolog protein-1 (NOTCH1) mutations in B- cell lymphocytic leukemia (B-CLL). Methods:A cohort of 105 Egyptian B-CLL patients aging from 43 to 86 years. PCR products including NOTCH1 exon 26, 27, and distal part of exon 34 expanding the sequences encoding transcription activation domain (TAD) and a peptide sequence rich in proline (P), glutamic acid (E), serine (S), threonine (T) (PEST domains) were sequenced by direct DNA Sanger sequencing. Results:NOTCH1 mutations were detected in 48/105 of patients (45.7%). Mutations in B-CLL patients are insertions (n=21), point mutations (n=18) and deletions (n=12). NOTCH1 mutations showed significant impact on prognosis of B-CLL patients as they were associated with increased bone marrow lymphocytes, more relapse and high incidence of mortality, shortened overall survival and progression free survival, and lymphocytes doubling time, when compared with NOTCH1 wild type B-CLL patients (P= 0.001; 0,005; 0.042; 0.049; 0.008; 0.049 respectively). Conclusion:NOTCH1 mutations were considered as bad prognostic marker in B-CLL and suggested to be included in risk stratification of B-CLL patients at diagnosis.

Identification of diterpenoid compounds that interfere with Fli-1 DNA binding to suppress leukemogenesis

The ETS transcription factor Fli-1 controls the expression of genes involved in hematopoiesis including cell proliferation, survival, and differentiation. Dysregulation of Fli-1 induces hematopoietic and solid tumors, rendering it an important target for therapeutic intervention. Through high content screens of a library of chemicals isolated from medicinal plants in China for inhibitors of a Fli-1 transcriptional reporter cells, we hereby report the identification of diterpenoid-like compounds that strongly inhibit Fli-1 transcriptional activity. These agents suppressed the growth of erythroleukemic cells by inducing apoptosis and differentiation. They also inhibited survival and proliferation of B-cell leukemic cell lines as well as primary B-cell lymphocytic leukemia (B-CLL) isolated from 7 patients. Moreover, these inhibitors blocked leukemogenesis in a mouse model of erythroleukemia, in which Fli-1 is the driver of tumor initiation. Computational docking analysis revealed that the diterpenoid-like compounds bind with high affinity to nucleotide residues in a pocket near the major groove within the DNA-binding sites of Fli-1. Functional inhibition of Fli-1 by these compounds triggered its further downregulation through miR-145, whose promoter is normally repressed by Fli-1. These results uncover the importance of Fli-1 in leukemogenesis, a Fli-1-miR145 autoregulatory loop and new anti-Fli-1 diterpenoid agents for the treatment of diverse hematological malignancies overexpressing this transcription factor.

Expression of the apoptosis-releated genes in patients with newly diagnosed chronic lymphocytic leukemia in clinical data context

Introduction.The given data of fundamental studies of apoptosis processes in B-cell lymphocytic leukemia (B-CLL) testifies about the complexity and variety of mechanisms affecting the kinetics of normal cells and tumor lymphocytes in this disease. It is important to study the severity of clinical manifestations of the disease depending on the expression of the genes that modulate apoptosis.The purposeof the study is to compare the activity of genes encoding apoptosis modulators, the cell cycle and cancer-testicular PRAME protein with clinical manifestations of the disease in primary patients with B-CLL.Materials and methods.The level of expression of the proapoptotic genes FAS, TRAIL, TNFR2, DR4/5 and DR3, as well as the HSP27, XIAP genes, blocking apoptosis was determined in 23 patients with newly diagnosed chronic B-CLL. In addition, expression of genes TP53 and P21 and cancer-testis gene PRAME are tested.Results.According to the multivariate regression analysis, the FAS gene expression in the onset of the disease had the greatest impact on the clinical characteristics of the disease. In this connection, the patients were divided into groups with normal (group) and low gene level (group II). A low level of FAS expression (Me 387 %) was associated with stage II disease (p = 0.03), a large number of lympho cytes (p = 0.001), fewer erythrocytes (p = 0.08), and a lower level of TNFR2 gene expression (p = 0.08), high level of expression of XIAP, HSP27, P21. Overall, the anti-apoptotic potential in Group II patients was higher, which was accompanied by more pronounced clinical manifestations of the disease.Conclusions.The increased anti-apoptotic potential of tumor lymphocytes in newly diagnosed B-CLL is accompanied by a larger tumor mass and greater clinical and hematological manifestation of the disease.

Highly similar genomic landscapes in monoclonal B-cell lymphocytosis and ultra-stable chronic lymphocytic leukemia with low frequency of driver mutations

Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we performed whole-genome sequencing in 11 individuals with monoclonal B- cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.

Abstract 2444: Recurrent mutations and clinical outcomes in patients with follicular lymphoma

Abstract Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma (NHL), is associated with a highly heterogeneous clinical course. The genetic landscape of FL is less understood than other B-cell NHLs such as chronic lymphocytic leukemia and diffuse large B-cell lymphoma. With ongoing clinical trials using therapies targeting B cell receptor signaling (e.g., ibrutinib) in FL, recurrent mutations could impact the interpretation of these trial outcomes and the identification of specific patient subpopulations that have increased or decreased clinical benefit. To identify novel recurrent mutations in FL, we designed a large custom capture lymphoma-related gene panel (WUSM-LP) and queried 113 predominantly treatment-naïve FL samples. To design the WUSM-LP reagent (NimbleGen), we targeted the coding regions of 1716 genes identified in exome sequencing data from a cohort of 24 patients (28 samples) with FL combined with genes recurrently mutated in B-cell non-Hodgkin lymphoma in the literature (Krysiak K et al., Blood, 2016). Previously, we applied the WUSM-LP to the discovery cohort and an additional 81, primarily treatment-naïve, FL samples. Using mutations identified in these 105 individuals, we identified 39 significantly mutated genes (SMGs). These included well-characterized genes in FL (e.g., KMT2D, EZH2, TNFRSF14), and identified novel mutations (BTK, HVCN1, and others) thereby expanding the repertoire of mutations affecting genes in the BCR signaling pathway, SWI/SNF complex and histone family. Including novel BTK mutations, we identified 23 (22%) individuals harboring mutations that likely up-regulate signaling downstream of BTK, potentially affecting response rates to BTK-targeting therapies at the point of FL diagnosis. We also focused on the subset FL patients (N=59) that were sequenced within 1 year of front-line therapy in order to identify clinical associations between recurrent mutations and clinical outcomes, comparing mutation status of SMGs to progression-free survival (PFS) rates. Improved PFS was observed in patients harboring HVCN1 mutations (p<0.05) in the treated cohort. CREBBP mutations were associated with significantly reduced PFS (p<0.05). Since our initial analysis, we have completed sequencing of an additional 174 FL samples with clinical data available. Confirming our earlier observations with an independent cohort, we observed 9 TNFAIP3 truncating, 18 CARD11 coiled-coil domain, 4 BCL10 truncating and 8 BTK mutations in these samples. These mutations affect 18.4% of FL patients in this cohort, are likely to up-regulate NFkappaB signaling and may impact ibrutinib sensitivity. With the additional power of 8 HVCN1 and 110 CREBBP mutations we have identified, we will evaluate the previously identified associations. The analyses of recurrent mutations and clinical correlations in this combined cohort of nearly 300 FL patients are ongoing and will be presented. Citation Format: Kilannin Krysiak, Cody Ramirez, Felicia Gomez, Christopher A. Miller, Robert S. Fulton, Friederike Kreisel, Amanda F. Cashen, Nancy L. Bartlett, Ana Ruano, Eric D. Hsi, Malachi Griffith, Obi L. Griffith, Todd A. Fehniger. Recurrent mutations and clinical outcomes in patients with follicular lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2444. doi:10.1158/1538-7445.AM2017-2444

Simultaneous Occurrence of B-Cell Lymphocytic Leukemia and Acute Myeloid Leukemia in an Elderly Female Patient

Introduction: The coexistence of B-cell chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) in the same patient is rare. The aim of this study is to report of simultaneous occurrence of CLL and AML M5 in an old female patient for the first time. Case Presentation: A 72-year-old woman referred to hematology clinic for evaluation of leukocytosis, anemia, and thrombocytopenia. She had no known history of chronic illness or exposure to radiation or myelotoxic drugs. Physical examination showed the existence of generalized lymphadenopathy and splenomegaly. Section of the biopsy core disclosed a diffuse infiltration of lymphoid cells with hyperchromic irregular nuclei and scant cytoplasm in the background. Immunohistochemical staining for CD20 in lymphoid cells and CD68 in blastic cells were positive but it was negative for terminal deoxynucleotidyl transferase (TdT). The flow cytometric immunophenotyping analysis was performed in the presence of CD5 positive B-cell population (CD19; CD20 dim; CD23 and CD5/CD19) with small cell size that comprised 76% of cells with abnormal phenotype was revealed. Simultaneous occurrence of CLL and AML M5 was confirmed in the patients. Conclusions: We report a case of previously untreated CLL and AML M5 with rapidly progressive course to death in less than one month from diagnosis. To the best of our knowledge, the development of AML M5 in patient with CLL has not been reported before.

T Cells Bearing Anti-CD19- and/or Anti-CD38-Chimeric Antigen Receptors Effectively Abrogate Primary Double-Hit Lymphoma Cells

Patients with B-cell lymphomas bearing MYC translocation combined with an additional translocation involving other genes, such as BCL2, BCL3, or BCL6, whose category is defined as double-hit lymphoma (DHL), have dismal prognosis, because these cells are refractory to conventional immunochemotherapy. Recent studies have expanded the concept of a such disease entity to include double-expressing lymphomas (DEL) that co-overexpress MYC protein with those proteins, which have also poor prognosis. An adoptive T-cell immunotherapy with a chimeric antigen receptor (CAR) is clinically shown to have a powerful cytotoxicity in refractory neoplasias. Especially, several results showed that T cells transduced with an anti-CD19-CAR have successfully worked well in patients with refractory acute B-cell lymphoblastic leukemia and B-cell lymphoma as well as chronic B-cell lymphocytic leukemia. Thus, CAR T-cell therapy is a clinically promising tool for various refractory hematopoietic disorders. Accordingly, we examined whether anti-CD19- and/or anti-CD38-CAR-T cells, both of which we previously developed, could abrogate DHL cells. Here, we revealed that the remarkable cytotoxicity of anti-CD19- and/or anti-CD38-CAR T cells against DHL cells from patients. Firstly, DHL cell line cells (KPU-H1, a generous gift form Dr. Junya Kuroda, Kyoto Prefectural University of Medicine) were co-cultured with anti-CD19- and/or anti-CD38-CAR T cells at an effector (E) target (T) ratio of 1: 2 for three days. Cells harvested and stained with anti-CD19 and/or anti-CD38 antibody-APC or -PE were subjected to flow cytometry. Flow cytometric analysis showed that anti-CD19- or anti-CD38-CAR T cells almost killed KPU-H1 cells, respectively (specific cytotoxicity was >90%). Intriguingly, T cells expressing anti-CD19-CAR exerted a collaborative cytotoxicity against KPU-H1 cells with anti-CD38-CAR T cells in vitro. CD38-specific T cells were co-cultured with cytogenetic DHL (n=3) or DEL (n=2) cells from five patients carrying a poor prognosis for 3 days. We examined whether T cells retrovirally transduced with anti-CD19- and/or anti-CD38-CAR vector could show cytotoxicity against primary DHL cells obtained from patients. Anti-CD19 and/or ant-CD38-CAR T cells were co-cultured with primary DHL cells at an E: T ratio of 1: 2 for 3 days. Interestingly, anti-CD19 and anti-CD38-CAR T cells completely abolished these DHL cells from patients, respectively. Additionally, anti-CD19- and anti-38-CAR T cells were synergistically effective to eliminate DHL cells. These results showed that DHL cells, which are refractory or resistant to existing chemotherapeutic agents, can be efficiently abrogated by a clinical use of T cells with anti-CD19- and/or anti-CD38-CAR. These results might warrant adoptive immunotherapy with autologous T cells transduced with anti-CD19 and anti-CD38-CAR for patients with refractory DHL. DisclosuresNo relevant conflicts of interest to declare.

Transformation of Canine Lymphoma/Leukemia to More Aggressive Diseases: Anecdotes or Reality?

Transformation is the evolution of an indolent lymphoma/leukemia to an aggressive lymphoma, typically harboring a very poor prognosis. This phenomenon is well described in humans, but underestimated in dogs although recognized as a possible evolution of indolent lymphomas/leukemias. In canine chronic leukemias, blast crisis (mainly in myeloid) and Richter syndrome (transformation into a high grade lymphoma) (mainly in B-cell lymphocytic leukemia) have been reported. Transformation is a possible event also in canine low grade lymphomas, although rare. The increased knowledge has also generated new questions and posed challenges that need to be addressed to improve outcome, including the recognition of the clinical characteristics at diagnosis associated with a higher risk of transformation in an attempt of anticipating the typical evolution.

Phosphorylation of serine 523 on 5-lipoxygenase in human B lymphocytes

The key enzyme in leukotriene (LT) biosynthesis is 5-lipoxygenase (5-LO), which is expressed in myeloid cells and in B lymphocytes. There are three phosphorylation sites on 5-LO (Ser271, Ser523 and Ser663). Protein kinase A (PKA) phosphorylates 5-LO on Ser523. In this report, we demonstrate by immunoblotting that native 5-LO in mantle B cell lymphoma (MCL) cells (Granta519, JEKO1, and Rec1) and in primary chronic B lymphocytic leukemia cells (B-CLL) is phosphorylated on Ser523. In contrast, we could not detect phosphorylation of 5-LO on Ser523 in human granulocytes or monocytes. Phosphorylated 5-LO was purified from Rec1 cells, using an ATP–agarose column, and the partially purified enzyme could be dephosphorylated with alkaline phosphatase. Incubation of Rec1 cells with 8-Br-cAMP or prostaglandin E2 stimulated phosphorylation at Ser523. Furthermore, FLAG-5LO was expressed in Rec1 cells, and the cells were cultivated in the presence of 8-Br-cAMP. The 5-LO protein from these cells was immunoprecipitated, first with anti-FLAG, followed by anti-pSer523-5-LO. The presence of 5-LO protein in the final precipitate further supported the finding that the protein recognized by the pSer523 antibody was 5-LO. Taken together, this study shows that 5-LO in B cells is phosphorylated on Ser523 and demonstrates for the first time a chemical difference between 5-LO in myeloid cells and B cells.

CSF1R Protein Expression in Reactive Lymphoid Tissues and Lymphoma: Its Relevance in Classical Hodgkin Lymphoma.

Tumour-associated macrophages (TAMs) have been associated with survival in classic Hodgkin lymphoma (cHL) and other lymphoma types. The maturation and differentiation of tissue macrophages depends upon interactions between colony-stimulating factor 1 receptor (CSF1R) and its ligands. There remains, however, a lack of consistent information on CSF1R expression in TAMs. A new monoclonal antibody, FER216, was generated to investigate CSF1R protein distribution in formalin fixed tissue samples from 24 reactive lymphoid tissues and 187 different lymphoma types. We also analysed the distribution of CSF1R+, CD68+ and CD163+ macrophages by double immunostaining, and studied the relationship between CSF1R expression and survival in an independent series of 249 cHL patients. CSF1R+ TAMs were less frequent in B-cell lymphocytic leukaemia and lymphoblastic B-cell lymphoma than in diffuse large B-cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma and cHL. HRS cells in cHL and, with the exception of three cases of anaplastic large cell lymphoma, the neoplastic cells in NHLs, lacked detectable CSF1R protein. A CSF1R+ enriched microenvironment in cHL was associated with shorter survival in an independent series of 249 cHL patients. CSF1R pathway activation was evident in the cHL and inactivation of this pathway could be a potential therapeutic target in cHL cases.