Malignant Lymphocytes Research Articles

The Immunomodulatory Mechanisms of BTK Inhibition in CLL and Beyond.

Bruton's tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a pivotal role in B cell biology and function. As an essential component of the B cell receptor (BCR) signaling pathway, BTK is expressed not only in B cells but also in myeloid cells, including monocytes/macrophages, dendritic cells, neutrophils, and mast cells. BTK inhibitors (BTKis) have revolutionized the treatment of chronic lymphocytic leukemia (CLL) and other B cell malignancies. Besides their well-characterized role in inhibiting BCR signaling, BTKis also exert significant immunological influences outside the tumor cell that extend their therapeutic potential and impact on the immune system in different ways. This work elucidates the immunomodulatory mechanisms associated with BTK inhibition, focusing on CLL and other clinical contexts. We discuss how BTK inhibition affects various immune cells, including B cells, T cells, and macrophages. The effects of BTKis on the profiles of cytokines, also fundamental parts of the tumor microenvironment (TME), are summarized here as well. This review also appraises the implications of these immunomodulatory actions in the management of autoimmune diseases and infections. Summarizing the dual role of BTK inhibition in modulating malignant lymphocyte and immune cell functions, this paper highlights the broader potential clinical use of compounds targeting BTK.

A-247 Targeting metabolic requirements of malignant lymphocyte migration and disease dissemination

A-247 Targeting metabolic requirements of malignant lymphocyte migration and disease dissemination

Large-scale genome-wide association studies reveal the genetic causal etiology between ankylosing spondylitis and risk of leukemia and lymphocytic malignancies.

Evidence from multiple observational studies suggests that ankylosing spondylitis (AS) is associated with leukemia and lymphocytic malignancies. However, the obtained results are inconsistent, and the causal relationship still needs to be determined. In this context, we utilized two-sample Mendelian randomization (MR) to investigate potential causal associations between AS and leukemia and lymphocytic malignancies. The analysis was conducted through published genome-wide association studies (GWAS). We obtained genetic data on AS as the exposure and leukemia, including lymphocytic leukemia, myeloid leukemia, and lymphocytic malignancies including lymphoma, multiple myeloma (MM) as the endpoint. The main method to evaluate causality in this analysis was the inverse variance weighting (IVW) technique. Additionally, we employed the weighted mode, weighted median, and MR-Egger regression for supplementary analyses. Finally, heterogeneity tests, sensitivity analyses, and multi-effect analyses are carried out. In a random-effects IVW analysis, we found that genetic susceptibility to AS was associated with an increased risk of leukemia (OR = 1.002; 95%CI, 1.001-1.003; p = 0.003) and an increased risk of lymphocytic leukemia [OR = 1.001; 95% CI, (1.000-1.002), p = 0.008]. There was no evidence that AS was associated with lymphoma, myeloid leukemia, and MM. Our research indicates that AS was associated with an elevated risk of leukemia, and further analysis of specific types of leukemia showed that the risk of lymphocytic leukemia was associated with AS. Our findings highlight the importance of active intervention and monitoring to mitigate leukemia, especially lymphocytic leukemia risk in patients with AS.

Disruption of the ZFP574–THAP12 complex suppresses B cell malignancies in mice

Despite the availability of life-extending treatments for B cell leukemias and lymphomas, many of these cancers remain incurable. Thus, the development of new molecular targets and therapeutics is needed to expand treatment options. To identify new molecular targets, we used a forward genetic screen in mice to identify genes required for development or survival of lymphocytes. Here, we describe Zfp574, an essential gene encoding a zinc finger protein necessary for normal and malignant lymphocyte survival. We show that ZFP574 interacts with zinc finger protein THAP12 and promotes the G1-to-S-phase transition during cell cycle progression. Mutation of ZFP574 impairs nuclear localization of the ZFP574-THAP12 complex. ZFP574 or THAP12 deficiency results in cell cycle arrest and impaired lymphoproliferation. Germline mutation, acute gene deletion, or targeted degradation of ZFP574 suppressed Myc-driven B cell leukemia in mice, but normal B cells were largely spared, permitting long-term survival, whereas complete lethality was observed in control animals. Our findings support the identification of drugs targeting ZFP574-THAP12 as a unique strategy to treat B cell malignancies.

Preliminary Experience in Ultra-High Frequency Ultrasound Assessment of Cutaneous Primary Lymphomas: An Innovative Classification.

Primary cutaneous lymphoma (PCL) is a rare form of extranodal non-Hodgkin's lymphoma characterized by malignant lymphocytes confined to the skin. Accurate diagnosis and staging are crucial for optimal management, yet radiological literature on imaging PCL remains limited. This study aims to delineate the imaging characteristics of PCLs using high and ultra-high frequency ultrasound (UHFUS) and proposes a classification system based on ultrasound findings. A cohort of 88 individuals with suspected PCL underwent high-resolution ultrasound (HRUS) and color Doppler examination of lesions. Lesions were categorized based on sonographic appearance, and subsequent histopathological assessment confirmed the diagnosis. Ultrasound imaging revealed distinct patterns for primary cutaneous T-cell lymphomas (PCTCL) and primary cutaneous B-cell lymphomas (PCBCL), with characteristic features such as hypoechoic nodules, pseudonodular lesions, and dermal infiltration. Histopathological analysis confirmed the ultrasound findings, supporting the proposed classification system. Ultrasonography, particularly UHFUS, offers valuable insights into the imaging characteristics of primary cutaneous lymphomas, aiding the accurate diagnosis and assessment of treatment response. The proposed classification system based on ultrasound findings enhances the diagnostic approach to PCLs, and paves the way for improved patient care and management strategies.

Optic nerve infiltration as a significant feature in the treatment of a rare secondary intraocular lymphoma derived from a primary breast B-cell lymphoma: A case report

Intraocular lymphoma (IOL) is a rare form of lymphocytic malignancy which may pose a significant diagnostic challenge as it may masquerade various uveitis entities, with secondary IOL being less common than primary IOL. A 47-year-old woman presented with blurring of the right eye and floaters since 5 months before admission. She had a history of complete remission from primary breast diffuse large B cell lymphoma for five consecutive years. Both eyes had vitreous haziness and several yellowish, thickened retinal lesions with edematous and infiltrated optic nerves. The best-corrected visual acuity was hand movement for the right eye and 6/45 for the left eye. Cytopathologic exam of the vitreous showed a B cell origin lymphoma. Cerebral magnetic resonance imaging revealed new parenchymal lesions suggestive of metastatic lesions. The patient was given multiple rituximab intravitreal injections and systemic chemotherapy. Treatment options for IOL might be influenced by the central nervous system (CNS) involvement, which is signified by infiltration of the optic nerve in this case.

Molecular Subtypes and the Role of TP53 in Diffuse Large B-Cell Lymphoma and Richter Syndrome.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy and a heterogeneous entity comprised of several biologically distinct subtypes. Recently, novel genetic classifications of DLBCL have been resolved based on common mutational patterns indicative of distinct pathways of transformation. However, the complicated and costly nature of the novel classifiers has precluded their inclusion into routine practice. In view of this, the status of the TP53 gene, which is mutated or deleted in 20-30% of the cases, has emerged as an important prognostic factor for DLBCL patients, setting itself apart from other predictors. TP53 genetic lesions are particularly enriched in a genetic subtype of DLBCL that shares genomic features with Richter Syndrome, highlighting the possibility of a subset of DLBCL arising from the transformation of an occult chronic lymphocytic leukemia-like malignancy, such as monoclonal B-cell lymphocytosis. Patients with TP53-mutated DLBCL, including those with Richter Syndrome, have a particularly poor prognosis and display inferior responses to standard chemoimmunotherapy regimens. The data presented in this manuscript argue for the need for improved and more practical risk-stratification models for patients with DLBCL and show the potential for the use of TP53 mutational status for prognostication and, in prospect, treatment stratification in DLBCL.

STAT3 Expression and Its Correlation with PD-L1 Expression in Non-Hodgkin's Lymphoma and Hodgkin's Lymphoma at Dr. Saiful Anwar Regional Public Hospital in Malang, Indonesian Population.

Lymphomas are malignant lymphocyte neoplasms that globally account for 10% of cancers in individuals aged <20 years. Malignant lymphomas are divided into Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Despite the availability of many therapeutic modalities for lymphoma, such as Brentuximab vedotin, Nivolumab, and Pembrolizumab, it is still necessary to identify appropriate strategies with minimal side effects. Immunotherapy is a promising approach, exemplified by targeting JAK/STAT3 signaling, which can inhibit tumor growth and enhance antitumor immune responses. Hence, STAT3 (signal transducer and activator of transcription 3) is a promising therapeutic target. PD-L1 (programmed death-ligand 1), an immune checkpoint molecule, is used as a frontline treatment for various cancers. This study aims to determine STAT3 expression and its correlation with PD-L1 expression in NHL and HL to serve as a basis for further research on anti-STAT3 and its combination with other therapy targets. Samples were obtained from paraffin blocks of patients with confirmed diagnoses of NHL and HL, and then immunohistochemical staining was carried out with PD-L1 and STAT3 antibodies. The collected data were then analyzed using SPSS. Among the 10 HL patients, no patients (0%) expressed STAT3, while nine patients (90%) expressed PD-L1. Among the 10 NHL patients, 1 patient (10%) expressed STAT3, while six patients (60%) expressed PD-L1. There were no significant differences in STAT3 expression and PD-L1 expression between HL patients and NHL patients. There was no correlation between STAT3 and PD-L1 expression in HL and NHL because almost all STAT3 expressions were negative. Although this study revealed no differences between STAT3 and PD-L1 expression in HL and NHL and no significant correlation between STAT3 and PD-L1 expression in HL and NHL, this may serve as the basis for understanding the role of STAT3 and PD-L1 in the regulation of HL and NHL, which may be useful for further research targeting STAT3 and PD-L1 immunotherapy in HL and NHL.

Primary Renal Lymphoma in a Cat

Background: Lymphoma is a hematopoietic tumor characterized by the clonal proliferation of malignant lymphocytes. It is the most frequently observed neoplasm in cats, accounting for approximately 33% of all tumors diagnosed in this species. Primary renal lymphoma accounts for approximately 1.5% of tumors diagnosed in felines and usually affects middle-aged to elderly cats and European breeds. This study describes a case of renal lymphoma in a feline, emphasizing the clinical, laboratory, imaging, and anatomopathological findings of this disease. Case: A 3-year-old spayed mixed breed cat, weighing 3.3 kg, presented apathy, lack of appetite, prostration, polydipsia, restlessness, tremors, sudden incoordination, and increased abdominal volume. Blood counts, serum biochemistry, urinalysis, radiography, and abdominal ultrasonography were performed. Blood count showed leukocytosis, with many atypical lymphocytes and thrombocytopenia. Serum biochemistry revealed azotemia, mild hypokalemia, and low alkaline phosphatase levels. Radiography showed an increase in the kidney dimensions and asymmetry of the kidney silhouettes, indicating a mass effect. Ultrasonography revealed enlarged dimensions, irregular contours, and bilateral nodular features. However, the corticomedullary definition was preserved. Kidney cytology revealed a high number of lymphocytes in smears, with a predominance of medium to large lymphocytes, suggestive of lymphoma. The animal underwent chemotherapy with lomustine, vincristine, prednisolone, and doxorubicin; however, due to the recurrence of clinical signs, the animal died. Macroscopically, the kidneys were enlarged, irregularly contoured, and white in color. When cut, a loss of renal architecture was noted, with blurring of the cortical and medullary regions. Histologically, the renal architecture was obliterated and replaced by a monomorphic population of atypical lymphocytes. The histological diagnosis was compatible with diffuse renal large-cell, high-grade, immunoblastic lymphoma. Immunohistochemistry showed positive immunostaining for CD20 and PAX-5 in the cell cytoplasm and negative immunostaining for CD3. Ki67 was detected in approximately 40% of neoplastic cells. The final diagnosis was primary diffuse large B-cell renal lymphoma. Discussion: The diagnosis of primary renal lymphoma was based on the occurrence of this tumor only in the kidneys of the animal. Renal lymphoma is the most common primary neoplasm affecting cat kidneys. The diagnosis of lymphoma in animals is based on cytological and/or histological examination. In addition, the final classification is performed using immunophenotyping (T and B cells), histology, and/or cytology to evaluate the cellular and nuclear characteristics, histological organization, and growth patterns of the lymphomas. The current clinical signs are related to renal failure, including neurological signs caused by uremia. There is a relationship between the occurrence of lymphoma in felines and retroviral infection; however, it was not possible to test the animal for retroviruses. The alterations observed in hematological examinations are related to the ongoing neoplastic process. The imaging findings in this case were similar to those observed in other studies on renal lymphoma. The treatment used in this study was the L-CHOP protocol, which has been associated with the treatment of different types of lymphomas in several studies on feline lymphomas. The median survival of cats with renal lymphoma is 50 days. In this case, the patient survived 30 days after hospital admission. Keywords: kidney, neoplasm, necrosis, immunohistochemistry.

Exploring the association of disease-modifying therapies for multiple sclerosis and BTK inhibitors with epilepsy.

Multiple lines of evidence suggest a role of inflammation in epilepsy. Seizure incidence in patients with multiple sclerosis (MS) is twofold to threefold higher than the age-matched general population. To explore the association of MS disease-modifying therapies (DMTs) and FDA-approved Bruton tyrosine kinase inhibitors (for lymphocytic malignancies) with the occurrence of epilepsy using the US Food and Drug Administration Adverse Event Reporting System (FAERS) database. Secondary analysis of the FAERS database. We conducted a disproportionality analysis of FAERS between 2003-Q4 and 2023-Q3. MS DMTs and the Bruton tyrosine kinase inhibitor, ibrutinib, were included in the analysis. An inverse association was defined by a 95% confidence interval (CI) upper limit of reporting odds ratio (ROR) <1. We found an inverse association of ibrutinib, ocrelizumab, ofatumumab, rituximab, and teriflunomide with epilepsy. The strongest inverse association was seen with ibrutinib (ROR: 0.338; 95% CI: 0.218-0.524). Our findings suggest the possibility of considering these medications for repurposing opportunities in epilepsy and support a potential pathogenic role of leukocyte subsets in seizure perpetuation.

Blinatumomab in Practice.

Acute lymphoblastic leukemia (ALL) is a rare hematologic neoplasm in adults, with most cases defined by pathology related to abnormal B cell proliferation known as B-cell ALL. The course is challenging, with less-than-optimal survival outcomes, even with aggressive multiagent chemotherapy and consideration for stem cell transplantation. Novel therapies focused on targetable pathways are being investigated to improve outcomes while simultaneously decreasing toxicity. In our review, we aim to evaluate the utilization of blinatumomab in B-cell ALL and provide insight on how this guides our management. Blinatumomab is a bispecific T-cell engager (BiTE) immunotherapy that neutralizes malignant cells by instigating CD3-positive T cells to target CD19-positive B cells. However, this therapy targets both malignant and non-malignant lymphocytes with potentially severe side effects such as cytokine release syndrome or neurotoxicity. Evidence evaluating utilization in the relapsed or refractory setting has been most supported; however, newer trials have also indicated improved survival in the frontline treatment of B-cell ALL. As this therapy is relatively new, the treatment team may include members who are less experienced with the typical treatment course and drug mechanics. This review synthesized available data investigating the effectiveness of blinatumomab effectiveness and its adverse events in addition to providing guidance on safe administration methods utilizing a multidisciplinary healthcare team. When care is coordinated in these settings, serious side effects can be recognized early, allowing for necessary intervention leading to improved quality of life and overall survival. Future research will continue to evaluate blinatumomab in different lines of therapy and expand its way into community settings.

Single Cell RNA Sequencing Enables Identification of Mantle Cell Lymphoma Subclones with Drug-Resistant Profiles Already at Diagnosis and May be Used for Therapy Prognostication and Personalization

Mantle cell lymphoma (MCL) is a chronically relapsing B cell lymphoproliferative malignancy characterized by translocation t(11;14)(q13;q32), cyclin D1 overexpression and significant chromosomal instability. One of the main hypotheses of MCL relapses is a clonal evolution driven by therapy selection of subpopulations carrying adverse genetic and epigenetic aberrations. Our previous whole exome sequencing analysis of 25 MCL tumors showed that the majority of the detected single-nucleotide variants and copy number variants (CNV) were already detectable at diagnosis. To further analyze the subclonal structure of MCL at diagnosis and clonal evolution at relapse, we implemented single cell RNA sequencing analysis of five patients with MCL at diagnosis and at the first clinical relapse after failure of standard immunochemotherapy. All analyzed patients achieved at least partial remission. Fresh-frozen tumor cells were sorted to obtain Hoechst − CD45 + population which was processed using single cell 3′ protocol (10X Genomics). The libraries were paired-end sequenced on Novaseq 6000 (Illumina). The reads were aligned to GRCh38 human genome. The MCL cells were distinguished from the normal B cells based on of CCND1 expression and unsupervised clustering. CNVs were predicted using inferCNV package. These CNVs shared a high degree of similarity with the CNVs predicted using available exome sequencing data. The t-SNE representation of the data clearly showed unique positions for the malignant lymphocytes obtained from each of the five analyzed patients ( Figure 1a). In contrast, non-malignant cells (T cells, B cells and monocytes) have grouped together irrespective of the patient or compartment of origin ( Figure 1b). These data suggest two conclusions. First, each patient's malignant cells have unique genetic background. Second, the impact of the microenvironment does not override the impact of the genetic makeup of the non-malignant cells. The data thus suggest comparability of the malignant cells obtained from different compartments. Hierarchical clustering of the per-cell CNV profiles revealed subclonal heterogeneity of the MCL population in all analyzed compartments both at diagnosis and at relapse. In all analyzed patients the clustering identified several large subclones with significant differences (DG L) and at least one minor subclone (DG S) with significant similarities compared to the major clone detectable at relapse (R L). These data suggest that the majority of the diagnostic MCL cells (i.e., DG L clones) were indeed eliminated by therapy, which on clinical level corresponded to achieving remission. On the other hand, the data also suggest that MCL cells with CNV profiles highly similar to the major relapse clone (i.e., R L) were detectable already at diagnosis (i.e., DG S, Figure 1c). To gain a better insight into the biology of the DG S clones with potential drug-resistant phenotype, we analyzed transcriptome differences between DG S and DG L clones. For each patient we identified different sets of significantly upregulated genes in DG S compared to DG L. These included genes coding for important cell surface molecules ( MARCKS, CD52, CD79A, LTB), regulators of cell motility and cytoskeleton dynamics ( STMN1, SWAP70, CFL1, TUBB, TUBA1B, TMSB4X), transcription factor PRDM2, energy metabolism proteins ( MT-ND4, MT-CO1, ATP5MG), protein degradation proteins ( UBA52, PSMC4, UFD1, UBE2L3) and others. Among these genes, significant upregulation of TMSB4X, SWAP70, HLA-A, TUBB4B, RPS26, UBA52, PSMB2, SNRPG, ARPC1B, ATP5MG was detected in three out of five patients. Many of these genes code for oncogenes involved in cell motility and aggressiveness. To further characterize subclonal clusters in MCL population we used marker gene detection and subsequent gene set enrichment analysis, which identified cell adhesion molecules as major category upregulated in four out of five patients in clusters associated with relapse. Our data shows the applicability of the single cell genomic approaches not only to analyze subclonal heterogeneity for the study of disease biology, but also to identify drug-resistant subclones at diagnosis, predict response to various types of therapies thereby enabling therapy personalization. Acknowledgement: Supported by Grant Agency of the Czech Republic GA23-05474S and National Institute for Cancer Research (EXCELES) LX22NPO5102.

The Unique BCR Inhibiting Properties of BMS-986205 in Chronic Lymphocytic Cells

B cell receptor (BCR) signaling is recognised as a central pathway in the pathogenesis of chronic lymphocytic leukaemia (CLL), the most common leukemia in the western world. In CLL, the BCR pathway is activated by antigens in the tissue microenvironment to promote the maintenance and expansion of leukemic cells. The impressive inhibition of clonal expansion of CLL cells and the disease control achieved with tyrosine kinase inhibitors that block BCR signaling provided further evidence of the role of BCR in CLL. Indoleamine 2,3-dioxygenase 1 (IDO1) is the inducible and rate-limiting enzyme involved in the catabolism of tryptophan to kynurenine. It was discovered as a modulator of the innate immune response during infection. Subsequent discoveries have implicated IDO1 as a player in acquired immune tolerance. IDO1 activity has been identified in several types of cancer cells as well as in their tumor-surrounding microenvironment. We have previously investigated and characterized the impact of IDO1 expression in CLL. Our results show that CLL microenvironmental stimuli positively modulate IDO1 expression in leukemic cells and that increased IDO1 activity leads to sustained survival of malignant lymphocytes and impaired drug sensitivity through the autocrine/paracrine activation of the aryl hydrocarbon receptor by kynurenine (Atene CG et al. 2022). To assess the functional and clinical significance of IDO1 enzymatic activity in CLL, we used several inhibitors that are currently in late-stage clinical trials. Using BMS-986205 (BMS), an irreversible inhibitor of IDO1 with the best cell-based potency, in primary patient cells, we observed not only a reduction in kynurenine production due to enzymatic inhibition of IDO1, but also lower levels of IDO1 RNA and protein following BCR stimulation with α-IgM. As we have previously shown that BCR activation can induce IDO1 expression, we treated CLL cells with increasing doses of BMS for 3 hours and then added α-IgM to the culture. BMS is able to counteract the pro-survival effect of BCR triggering, leading to significant apoptosis of CLL cells, while the viability of CLL cells in the absence of external stimuli was not significantly modulated. Focusing on the complex signaling cascade activated by BCR engagement, we observed that BMS impaired the phosphorylation of upstream kinases such as SYK and BTK, but also CD79a, CD19 and BLNK, in addition to downstream effectors such as AKT. We therefore investigated which factors that negatively regulate the BCR pathway are modulated by BMS treatment. By analyzing both mRNA and protein levels, we measured a significant induction of the phosphatases PTPN6, PTPN22 and SHIP1. Since BMS also showed a downregulation of total SYK expression, we are analyzing the expression of the proto-oncogene CBL, an E3 ubiquitin ligase known to be involved in the negative regulation of SYK. Further studies should be undertaken to determine whether, in addition to its role as an IDO1 inhibitor, BMS may also have the ability to indirectly modulate and disrupt BCR signaling. Efforts are underway to identify the proteome-wide direct target of BMS in CLL cells. In conclusion, our limited and preliminary data suggest that BMS could be considered as a potential therapeutic strategy to target CLL cells with a double hit: by blocking the pro-survival effect of BCR signaling, one of the driving forces for CLL cell survival and proliferation, and by inhibiting IDO1/kynurenine action to restore sensitivity to key leukemia treatments.

A Phase I/II Study to Investigate the Addition of Venetoclax to Dasatinib and Steroids in Patients with Newly Diagnosed or Relapsed Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL): The Venda Trial, in Progress

Background: Acute Lymphoblastic Leukemia (ALL) is a hematologic malignancy of immature lymphocytes of either B cell or T cell origin. One particularly high -risk subset is Ph+ ALL, characterized by a translocation of chromosome 9 and 22, which generates the constitutively active BCR-ABL1 fusion tyrosine kinase capable of driving proliferation and survival of leukemic cells. Currently treatment options include multi-agent chemotherapy along with an ABL-specific tyrosine kinase inhibitor (TKI), though these regimens can lead to significant toxicity, particularly in older adults. Venetoclax is a potent and selective small molecule inhibitor of the B-cell lymphoma 2 (BCL-2) anti-apoptotic protein. Strong mechanistic and preclinical data have demonstrated strong synergistic activity between dasatinib and venetoclax I xenograft models and ex vivo patient samples, and provide an impetus to further explore the combination of BCR-ABL and BCL-2 inhibition in vivo. Based on these data, we designed a clinical trial to investigate the safety and preliminary efficacy of venetoclax in combination with the second generation TKI dasatinib in Ph+ ALL. Study Design and Methods This is an open label, Phase I/II investigator initiated trial evaluating the addition of venetoclax to dasatinib and steroids (with Rituximab in CD20+ patients). Phase I is the safety and dose-finding portion of the trial combining the BCL2 inhibitor venetoclax with dasatinib and steroids during the first 28 days of induction followed by dasatinib and venetoclax for the final 48 days of induction in newly diagnosed and relapsed Ph+ ALL and mixed phenotype acute leukemia (MPAL) patients, including patients with molecular only relapse. Primary inclusion criteria include a histologically confirmed diagnosis of Ph+ ALL or MPAL. Newly diagnosed Ph+ ALL or MPAL patients may not have received leukemia-directed therapy other than for steroids or hydrea, and relapsed patients must have not had prior dasatinib exposure. Key exclusion criteria include subjects with CML in myeloid blast crisis or Ph+ AML, ECOG &amp;lt; 2 and patients with a history of pleural effusions. n order to determine the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D), a Bayesian toxicity probability interval design applied to a pre-specified targeted dose limiting toxicity rate of 30%. Three dose cohorts investigating increasing doses of venetoclax will be enrolled. Once the RP2D dose has been identified, a Phase II expansion will examine preliminary efficacy of the combination. The primary endpoint is the rate of measureable residual disease (MRD) negative remission rate as assessed by both BCR-ABL RT-PCR and next generation sequencing (NGS) after induction. Secondary endpoints include relapse-free survival (RFS) and overall survival (OS). Correlative studies include BH3 profiling (measure of apoptosis) and ex vivo screening to dasatinib and venetoclax alone and in combination. Consolidation phase with blinatumomab plus dasatinib and venetoclax is planned for the cohort expansion group. Enrollment began in September 2022 and is expected to be complete by September 2025. This trial is registered at clinicaltrials.gov (NCT04872790).

Effect of graphene oxide nanoparticles on apoptosis of T-lymphocytes and Jurkat cells

Graphene and its derivatives are materials with unique physicochemical properties. A detailed study of these materials allows to consider them prospective biomedical agents for targeted drug and gene delivery, photothermal therapy of cancer, bioimaging, etc. However, this requires a comprehensive studies of their effects on the body tissues, including cells of the immune system.&#x0D; The aim of our research was to stydy the effects of nanoparticles based on pegylated graphene oxide (GO) upon apoptosis of T lymphocytes derived from blood of healthy donors and Jurkat 5332 cell line. Comparison of these cells will extend our knowledge of the effects of nanomaterials on the cells, and to respond the question, what results obtained with continuous cell lines are valid for normal non-malignant cells. In this work, we used GO nanoparticles (100-200 nm, 1-5 m) coated with linear (LP-GO) and branched (BP-GO) polyethylene glycol (PEG). The cells were cultured for 24 hours at 37 C and 5% CO2with nanoparticles at concentrations of 5 and 25 g/mL. Viability and early and late apoptosis of incubated Jurkat cells and CD3+cells from healthy donors were assessed by flow cytometry. It was found that the small nanoparticles coated with linear PEG at high concentrations (25 g/mL) could significantly reduce the number of live cells and increase the number of cells in late apoptosis. At the same time, large nanoparticles coated with branched PEG at high concentrations (25 g/mL) increased the percentage of T cells in early apoptosis.&#x0D; Meanwhile, the GO nanoparticles at both concentrations did not affect the viability and apoptosis of Jurkat cells, regardless of the size, concentration, and type of surface function of the particles.&#x0D; The obtained results suggest that GO nanoparticles exert different effects upon normal and malignant lymphocytes of T lineage. One may assume that these discrepancies could be explained by greater resistance of tumor cells compared to normal T cells. These findings suggests that studies of nanomaterials upon living cells should not be limited to experiments on cell lines, since their properties may significantly differ from those of non-malignant cells.

Hybrid Models Based on Fusion Features of a CNN and Handcrafted Features for Accurate Histopathological Image Analysis for Diagnosing Malignant Lymphomas.

Malignant lymphoma is one of the most severe types of disease that leads to death as a result of exposure of lymphocytes to malignant tumors. The transformation of cells from indolent B-cell lymphoma to B-cell lymphoma (DBCL) is life-threatening. Biopsies taken from the patient are the gold standard for lymphoma analysis. Glass slides under a microscope are converted into whole slide images (WSI) to be analyzed by AI techniques through biomedical image processing. Because of the multiplicity of types of malignant lymphomas, manual diagnosis by pathologists is difficult, tedious, and subject to disagreement among physicians. The importance of artificial intelligence (AI) in the early diagnosis of malignant lymphoma is significant and has revolutionized the field of oncology. The use of AI in the early diagnosis of malignant lymphoma offers numerous benefits, including improved accuracy, faster diagnosis, and risk stratification. This study developed several strategies based on hybrid systems to analyze histopathological images of malignant lymphomas. For all proposed models, the images and extraction of malignant lymphocytes were optimized by the gradient vector flow (GVF) algorithm. The first strategy for diagnosing malignant lymphoma images relied on a hybrid system between three types of deep learning (DL) networks, XGBoost algorithms, and decision tree (DT) algorithms based on the GVF algorithm. The second strategy for diagnosing malignant lymphoma images was based on fusing the features of the MobileNet-VGG16, VGG16-AlexNet, and MobileNet-AlexNet models and classifying them by XGBoost and DT algorithms based on the ant colony optimization (ACO) algorithm. The color, shape, and texture features, which are called handcrafted features, were extracted by four traditional feature extraction algorithms. Because of the similarity in the biological characteristics of early-stage malignant lymphomas, the features of the fused MobileNet-VGG16, VGG16-AlexNet, and MobileNet-AlexNet models were combined with the handcrafted features and classified by the XGBoost and DT algorithms based on the ACO algorithm. We concluded that the performance of the two networks XGBoost and DT, with fused features between DL networks and handcrafted, achieved the best performance. The XGBoost network based on the fused features of MobileNet-VGG16 and handcrafted features resulted in an AUC of 99.43%, accuracy of 99.8%, precision of 99.77%, sensitivity of 99.7%, and specificity of 99.8%. This highlights the significant role of AI in the early diagnosis of malignant lymphoma, offering improved accuracy, expedited diagnosis, and enhanced risk stratification. This study highlights leveraging AI techniques and biomedical image processing; the analysis of whole slide images (WSI) converted from biopsies allows for improved accuracy, faster diagnosis, and risk stratification. The developed strategies based on hybrid systems, combining deep learning networks, XGBoost and decision tree algorithms, demonstrated promising results in diagnosing malignant lymphoma images. Furthermore, the fusion of handcrafted features with features extracted from DL networks enhanced the performance of the classification models.

Identification and confirmation via in situ hybridization of Merkel cell polyomavirus in rare cases of posttransplant cutaneous T-cell lymphoma.

Viral infection is an oncogenic factor in many hematolymphoid malignancies. We sought to determine the diagnostic yield of aligning off-target reads incidentally obtained during targeted hematolymphoid next-generation sequencing to a large database of viral genomes to screen for viral sequences within tumor specimens. Alignment of off-target reads to viral genomes was performed using magicBLAST. Localization of Merkel cell polyomavirus (MCPyV) RNA was confirmed by RNAScope in situ hybridization. Integration analysis was performed using Virus-Clip. Four cases of post-cardiac-transplant folliculotropic mycosis fungoides (fMF) and one case of peripheral T-cell lymphoma (PTCL) were positive in off-target reads for MCPyV DNA. Two of the four cases of posttransplant fMF and the case of PTCL showed localization of MCPyV RNA to malignant lymphocytes, whereas the remaining two cases of posttransplant fMF showed MCPyV RNA in keratinocytes. Our findings raise the question of whether MCPyV may play a role in rare cases of T-lymphoproliferative disorders, particularly in the skin and in the heavily immunosuppressed posttransplant setting.

Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

It is well established that inflammation and platelets promote multiple processes of cancer malignancy. Recently, platelets have received attention for their role in carcinogenesis through the production of microvesicles or platelet-derived microparticles (PMPs), which transfer their biological content to cancer cells. We have previously characterized a new subpopulation of these microparticles (termed mito-microparticles), which package functional mitochondria. The potential of mitochondria transfer to cancer cells is particularly impactful as many aspects of mitochondrial biology (i.e., cell growth, apoptosis inhibition, and drug resistance) coincide with cancer hallmarks and disease progression. These metabolic aspects are particularly notable in chronic lymphocytic leukemia (CLL), which is characterized by a relentless accumulation of proliferating, immunologically dysfunctional, mature B-lymphocytes that fail to undergo apoptosis. The present study aimed to investigate the role of PMPs on CLL metabolic plasticity leading to cancer cell phenotypic changes. CLL cell lines were co-incubated with different concentrations of human PMPs, and their impact on cell proliferation, mitochondrial DNA copy number, OCR level, ATP production, and ROS content was evaluated. Essential genes involved in metabolic-reprogramming were identified using the bioinformatics tools, examined between patients with early and advanced CLL stages, and then validated in PMP-recipient CLLs. Finally, the impact of the induced metabolic reprogramming on CLLs' growth, survival, mobility, and invasiveness was tested against anti-cancer drugs Cytarabine, Venetoclax, and Plumbagin. The data demonstrated the potency of PMPs in inducing tumoral growth and invasiveness in CLLs through mitochondrial internalization and OXPHOS stimulation which was in line with metabolic shift reported in CLL patients from early to advanced stages. This metabolic rewiring also improved CLL cells' resistance to Cytarabine, Venetoclax, and Plumbagin chemo drugs. Altogether, these findings depict a new platelet-mediated pathway of cancer pathogenesis. We also highlight the impact of PMPs in CLL metabolic reprogramming and disease progression.

Abstract 380: BaF3-BTK engineering cell lines as a useful platform for novel BTK inhibitor discovery

Abstract Introduction: Bruton's tyrosine Kinase (Btk) plays an essential role in BCR signaling and has been a hot new target in molecular targeted therapy in the past few years, led by the clinical success of ibrutinib and acalabrutinib, two first approved BTK inhibitors, that greatly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Currently, there are 5 approved BTK inhibitors including additionally zanubrutinib, orelabrutinib and velexbru. Up to 24 BTK inhibitors are under active clinical evaluation as antineoplastic drugs against chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B-cell malignancies and mantle cell lymphoma (MCL) in different countries. However, the acquired resistance due to the C481S mutation in BTK remains an essential challenge to improve the poor prognosis of the patient. Subsequently, several next-generation BTK inhibitors are under clinical evaluation targeting the BTK-C481S mutation, such as LOXO-305, a highly selective and non-covalent BTK inhibitor which potently inhibits the cellular activity of BTK C481S/T/R mutations. Methods: Our group has generated 9 Ba/F3-BTK engineering cell lines as useful cell line and xenograft models for novel BTK inhibitor discovery and development. They harbor wild type BTK or ibrutinib resistance BTK mutations such as the C481S and C481S-double mutations, and the T474M/I mutations. Moreover, we generated Ba/F3-ETV6-BTK-F517L and Ba/F3-ETV6-BTK-C481S-F517L cell lines using a dose-escalation method after treating cells with a mutagenic agent. The BTK-F517L mutation is highly resistant to both ibrutinib and LOXO-305 treatment. Crystal structure analysis and 3D modeling revealed that the F517L mutation may affect the conformation of the BTK binding pocket to ibrutinib, indicating novel BTK inhibitors may be needed to overcome this resistance mutation. Conclusion: The Ba/F3-BTK engineering cell lines can be a useful tool to develop and evaluate next-generation BTK inhibitors and to explore new acquired resistance BTK mutations. Citation Format: Guoqian Wang, Hongling Chen, Yu Wang, Jingxiao Xu, Lin Zhou, Yao Tang, Jinying Ning, Feng Hao. BaF3-BTK engineering cell lines as a useful platform for novel BTK inhibitor discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 380.

Management of limited stage Hodgkin lymphoma

Hodgkin lymphoma (HL) is a lymphoid neoplasm characterized by malignant lymphocytes, known as Reed-Sternberg cells, on a background of non-neoplastic inflammatory cells. Lugano staging determines the stage of Hodgkin lymphoma, which, in turn, determines the treatment and prognosis. Limited-stage disease is defined as Stage I and Stage II, which is diagnosed in more than 50% of patients. Pre-treatment risk stratification, PET-adapted therapy, and combined modality treatment have significantly improved cure rates, making limited-stage HL one of the most curable malignancies. In this article, we discuss the current approach to managing limited-stage HL.