Indolent Chronic Lymphocytic Leukemia Research Articles

Revealing heterogeneity in chronic lymphocytic leukemia: AI-driven insights into aggressive and indolent disease subtypes.

e19029 Background: Chronic lymphocytic leukemia (CLL) exhibits a broad spectrum of clinical behaviors, from indolent courses requiring minimal intervention to aggressive forms demanding immediate treatment. The underlying heterogeneity of CLL poses significant challenges in predicting disease progression and tailoring patient-specific therapeutic strategies. Leveraging advanced and novel artificial intelligence (AI) and machine learning (ML) methodologies, our study aims to dissect the complexity of CLL, focusing on the identification and characterization of biomarkers that differentiate aggressive and indolent disease forms. Methods: The NetraAI is a priorietaryAI/ML system that is also being utilized in a research collaboration at a leading American oncology institute (not disclosed) that integrates machine intelligence, augmented intelligence, and an agent that interacts with AI-derived patient representations. This system was designed to learn from datasets with a small number of samples and many independent variables. This system operates by generating hypotheses in the form of interactive representations of patient populations that reveal heterogeneity along with statistically significant driving factors. Finally, an automated agent called AutoPlay interacts with these representations and generates an accounting of all variables, subpopulations of samples, and it also generates a human readable perspective on what was discovered by the data set. Results: We applied the NetraAI to a CLL dataset and used it to refine the analysis and interpretation of the corresponding transcriptomic data (GSE39411). Significantly lower levels of FADS3, GSDME, LPL, IMMT, NMB, and AEBP1 and higher expressions of COBLL1, P2RY1R, PDE8A, SYNE2, and FCRL3 were identified as hallmarks of indolent CLL, suggesting these markers could inform less aggressive disease management strategies. Furthermore, within a total of 104 CLL samples, we uncovered two distinct subpopulations within aggressive CLL, delineated by unique genetic markers. Group A, encompassing 31 aggressive CLL samples is predominantly characterized by the expression of lipoprotein lipase (LPL), while Group B, comprising 22/23 aggressive samples, is characterized by ZBTB20 and SYNE2. Our poster will also provide glimpses into a more refined heterogeneity that emerged from the analysis. Our results not only underscore the heterogeneity within aggressive CLL, but also highlights the potential for these markers to guide prognostic assessments and therapeutic decisions. Conclusions: This study not only advances our understanding of CLL heterogeneity, but also sets the stage for the development of more personalized and effective treatment modalities. By identifying drivers of disease aggression and indolence, we pave the way for targeted therapies that could significantly improve patient outcomes in CLL.

A Phase 1, Open-Label, Multicenter, Dose-Escalation Study of Sgr-1505 As Monotherapy in Subjects with Mature B-Cell Malignancies

Background and Significance: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Furthermore, a MALT1 inhibitor (JNJ-67856633) showed efficacy in mature B cell malignancies from phase 1 studies (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3) ABC-DLBCL cell lines. SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition may expand therapeutic options for patients with selected B-cell lymphomas, supporting further evaluation of SGR-1505 in clinical trials. Study Design and Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies (figure 1). At present, the study is open to accrual at multiple investigative sites in the US with plans to expand to Europe. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1-6 subjects, and at higher dose levels using a conventional 3+3 design.

Progressive Gut Microbiome Dysbiosis Is Associated with Chronic Lymphocytic Leukemia Pathogenesis and Development in the Eµ-TCL1 Mouse Model

Introduction: The gut microbiome is home to a community of microorganisms such as bacteria, fungi, viruses, archaea, and protozoa. Maintaining a balance between “favorable” and “unfavorable” bacteria within the gut microbiome is essential for homeostatic physiological processes including proper immune system development, regulation of metabolic pathways, protection against pathogens, and gut epithelium integrity. Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by the accumulation of CD5 + B-cells in the blood, bone marrow, and secondary lymphoid tissues. The pathogenesis of CLL is related to the activation of critical survival pathways such as B-cell and toll-like receptor signaling pathways, where microbial antigens are known to be key stimulators driving CLL B-cell proliferation. In this study, we investigated how the gut microbiota composition contributes and correlates to CLL disease progression. Methods: Using a well-established murine model representative of CLL development in humans, we profiled the gut microbiota of transgenic Eµ-TCL1 mice and age-matched wild-type B6 (WT B6) mice over the course of 12 months (n = 7-10/group). Fecal pellets were collected aseptically from study mice at four time points (4, 7, 10, and 12 months) for DNA extraction using the QIAamp® PowerFecal® Pro DNA Kit. To characterize the gut microflora, we utilized 16S rRNA sequencing using the Illumina 16S metagenomics protocol. Bacterial taxonomy was assigned and analyzed using the QIIME2™ platform. Linear Discriminant Analysis (LDA) Effect Size (LEfSe) analysis was employed to identify differentially expressed bacterial taxa between cohorts. Beta diversity indices were used to evaluate intersample differences in the fecal microbiota. Mice were monitored for peripheral expansion of leukemia cells (%CD45 +/CD19 +/CD5 + cells) via flow cytometry. Next, we evaluated the microbiome in an aggressive model of CLL. Utilizing the Eµ-TCL1 adoptive transfer murine model, similar methods were adopted to profile the gut microbiome over the course of 7 weeks. Lastly, to evaluate the contribution of the gut microbiota to CLL development, we employed an antibiotic-mediated gut microflora ablation to the adoptive transfer Eµ-TCL1 model, with leukemia expansion monitored post-engraftment on a weekly basis. Results: A unique microbiome signature was identified in both the indolent and aggressive model of CLL. Evaluation of beta diversity, using Bray-Curtis distance metrics, confirmed distinct differences in the gut microbial communities between Eµ-TCL1 and WT B6 cohorts. At the phylum level, the gut microbiota of the Eµ-TCL1 and WT B6 cohorts were similarly dominated by Firmicutes and Bacteroidota. However, Proteobacteria Deferribacterota and Desulfobacterota were more abundant in the Eµ-TCL1 mice over 12 months as compared to age-matched WT B6. Abundance of Actinobacteriota increased specifically at 12 months in the Eµ-TCL1 mice. Additionally, LEfSe analysis showed enrichment of certain phyla in the Eµ-TCL1 mice at 4 months, 10 months, and 12 months. Presence of these bacteria paralleled with expansion of disease. In the aggressive model of CLL, beta diversity confirmed distinct differences in gut microbial communities as CLL disease progressed. Moreover, certain phyla were enriched in leukemic mice at 7 weeks post-engraftment including Proteobacteria and Actinobacteriota. Lastly, in the antibiotic-mediated gut flora ablation model, a delay of disease onset was reflected in mice with ablated gut microbiomes compared to mice with intact gut microbiomes, resulting in prolonged overall survival. Conclusion: We provide novel results associating the gut microbiome with the initiation and progression of CLL disease. In both the indolent and aggressive CLL disease models, we showed that there is a distinct gut microbiome as evident by beta diversity. A dysbiotic signature represented by increasing Proteobacteria abundance was similarly identified in both models. Proteobacteria are known to be evident in diseases characterized by inflammation. These findings support future studies to determine a potential causal mechanism between gut microbiota changes and CLL pathogenesis.

ZNF683 marks a CD8+ Tcell population associated with anti-tumor immunity following anti-PD-1 therapy for Richter syndrome.

Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory Tcells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key Tcell genes (TCF7, LMO2, CD69) and impact pathways of Tcell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683high Tcells with response.

SGR‐1505‐101: A PHASE 1, OPEN‐LABEL, MULTICENTER, DOSE‐ESCALATION STUDY OF SGR‐1505 AS MONOTHERAPY IN SUBJECTS WITH MATURE B‐CELL MALIGNANCIES

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of the nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. In particular, constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity, and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. Aims: These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential and may expand therapeutic options for patients with selected B-cell lymphomas supporting further evaluation of SGR-1505 in clinical trials. Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies. At present, the study has been activated in 3 sites in the United States. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1–6 subjects, and at higher dose levels using a conventional 3+3 design. Encore Abstract—previously submitted to EHA 2023 The research was funded by: Schrodinger Keywords: aggressive B-cell non-Hodgkin lymphoma, molecular targeted therapies, ongoing trials Conflicts of interests pertinent to the abstract. B. Yoo Employment or leadership position: Schrodinger Stock ownership: Schrodinger Z. Nie Employment or leadership position: Schrodinger Stock ownership: Schrodinger G. Krilov Employment or leadership position: Schrodinger Stock ownership: Schrodinger J. Tan Employment or leadership position: Schrodinger Stock ownership: Schrodinger H. Wright Employment or leadership position: Schrodinger Stock ownership: Schrodinger D. Weiss Employment or leadership position: Schrodinger Stock ownership: Schrodinger W. Yin Employment or leadership position: Schrodinger Stock ownership: Schrodinger K. Akinsanya Employment or leadership position: Schrodinger Stock ownership: Schrodinger

How We Manage Patients with Indolent B-Cell Malignancies on Bruton’s Tyrosine Kinase Inhibitors: Practical Considerations for Nurses and Pharmacists

The most common forms of B-cell malignancy, non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), have seen a drastic shift in the treatment landscape over the last two decades with the introduction of targeted agents. Among them are Bruton's tyrosine kinase (BTK) inhibitors, which have demonstrated excellent efficacy in indolent B-cell NHLs and CLL. Although BTK inhibitors are generally thought to be more tolerable than chemoimmunotherapy, they are associated with a unique safety profile including varying rates of rash, diarrhea, musculoskeletal events, cardiovascular events, and bleeding. Ibrutinib was the first BTK inhibitor to gain a Health Canada indication, followed by second-generation BTK inhibitors acalabrutinib and zanubrutinib, which have better safety profiles compared to ibrutinib, likely due to their improved selectivity for BTK. As BTK inhibitors are oral agents given continuously until disease progression, long-term adverse event (AE) monitoring and management as well as polypharmacy considerations are important for maintaining patient quality of life. This paper intends to serve as a reference for Canadian nurses and pharmacists on dosing, co-administration, and AE management strategies when caring for patients with indolent B-cell NHL or CLL being treated with BTK inhibitors.

A comprehensive assessment of lymphocyte subsets, their prognostic significance, and changes after first-line therapy administration in patients with chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), changes in the peripheral blood lymphocyte subsets play an important role in disease progression and infectious complications. The impact of chemoimmunotherapy (CIT) on these changes has not been extensively studied METHODS: We used multi-color flow cytometry, to prospectively measure absolute and relative numbers of CD4+ and CD8+ T-cells and their subsets in 45 patients with indolent untreated CLL, 86 patients indicated for first-line treatment, and 34 healthy controls. In 55 patients, we analyzed the impact of CIT RESULTS: CLL patients had a significant increase in most cell populations in comparison to controls. Progression of CLL was characterized by significantly elevated counts with the exception of a lower percentage of naïve T-cells. After treatment, the percentage of naïve T-cells further decreased at the expense of effector memory T-cells (TEM). In patients with indolent CLL, higher percentages of naïve CD4+ (p= 0.0026) and naïve CD8+ (p= 0.023) T-cells were associated with a longer time to first treatment (TTFT). The elevation of CD4+ central memory T-cells (TCM) (p= 0.27) and TEM (p= 0.003) counts and a higher percentage of CD4+ TEM (p= 0.0047), were linked with shorter TTFT. In treated patients, increased regulatory T-cells count was associated with shorter time to next treatment (TTNT) (p= 0.042), while higher CD4+ TCM count with shorter TTNT (p= 0.035) and shorter overall survival (p= 0.041). Our results indicate that naïve cell depletion and CD4+ TCM and TEM increases are detrimental to CLL patients' prognosis.

ZNF683 (Hobit) Marks a CD8+ T Cell Population Associated with Anti-Tumor Immunity Following Anti-PD-1 Therapy for Richter Syndrome

Richter syndrome (RS), an aggressive B cell lymphoma originating from transformation of indolent chronic lymphocytic leukemia (CLL), has been shown to be responsive to PD-1 checkpoint blockade (CPB) across clinical trials. To systematically discover the determinants of this response, we analyzed single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 RS patients enrolled in a phase I study of the anti-PD1 drug nivolumab with concurrent ibrutinib (NCT 02420912). At pre-treatment sampling, RS responders (RS-R) had a larger CD8 effector/effector memory (E/EM) population than RS non-responders (RS-NR) (p=0.04, 2-sample t-test). This population was rare in normal bone marrow (N=30, p=0.001, 2-sample t-test), displayed intermediate exhaustion, preserved cytotoxicity and was marked by expression of the transcription factor and PRDM1-homolog, ZNF683. Further implicating ZNF683-expressing CD8 T cells in CPB response, comparison of gene expression between RS-Rs and RS-NRs within transcriptionally defined CD8 T cell clusters identified higher ZNF683 expression in RS-Rs. Lastly, the kinetics of ZNF683 expression correlated with response, with relative expression per cell decreasing with RS progression. Trajectory analysis supported a T cell differentiation path from GZMK+ memory to ZNF683intermediate followed by branching towards either ZNF683high or terminal exhaustion. To establish the extent to which ZNF683high T cell signatures are detectable in a broader population of RS patients, we examined bulk RNA-seq data generated from 35 independent RS biopsy specimens and found that 7 of 35 (20%) displayed a ZNF683high signature. A similar distribution of samples with ZNF683high expression, when corrected for T cell fraction, was observed in 18 of 81 (20%) CLL samples for which non-CD19 selected transcriptomic data was available and was associated with a trend towards improved overall survival (p=0.11). We also detected a similar ZNF683high signature in a large external compendium of single cell data from tumor infiltrating lymphocytes across 18 solid and 3 hematologic tumor types and in the peripheral blood of melanoma CPB responders (p<0.001 hypergeometric test). Upon evaluation of TCGA data, we detected an association with survival in melanoma cases with high T cell expression of ZNF683 (P<0.0001). To probe the cellular impact of ZNF683, a FLAG-tagged ZNF683 construct was overexpressed in Jurkat cells. ZNF683 overexpression revealed upregulation of PRF1, ITGA1, CD244, CD226 and IL2RB and downregulation of PRDM1 and IL2. CUT&RUN was performed to identify the binding sites of ZNF683, yielding twenty-four differentially regulated peaks, among which included ENCODE-identified regulatory regions adjacent to key immune genes (TNFAIP8, BTN3A2, CD69/KLRF1, ARHGAP2, CAMK4, LMO2, IL2, TCF7). Evaluation of external ATAC-seq data generated from exhausted T cell subsets revealed PD-1high tumor-infiltrating lymphocytes to have reduced accessible chromatin across identified putative ZNF683 binding sites, including TCF7. Thus, terminal exhausted cells not only lose ZNF683 expression but also display chromatin remodeling that likely abrogates its effects. By applying the tool CISTROME-GO to integrate the CUT&RUN and RNA-seq data, we observed enrichment in pathways corresponding to antigen binding and presentation, transcription factor activity, and T cell mediated cytotoxicity and cell killing upon ZNF683 expression. To determine if the association between ZNF683 expression and response to PD-1 blockade in RS could be confirmed in peripheral blood, we analyzed RNA-seq data generated from T cells isolated from pre-treatment blood samples from 7 independent RS patients treated with PD-1 therapy. We again identified ZNF683 among the top upregulated genes in RS-R (Log2 fold change = 2.13, p=0.037), along with other genes enriched in our ZNF683high CD8 E/EM signature (BATF, CORO1A, CD38, ITGB2, GZMM), while RS-NR were enriched in NK-like genes (KLRC1, FCGR3A, KLRG1, KLRF1 and FCER1G). In conclusion, we identify ZNF683 as marking a CD8 T cell population associated with CPB response in RS blood and bone marrow and provide evidence that this transcription factor regulates key cellular pathways involved in immune anti-tumor response, with implications for understanding CPB response in RS and other malignancies.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

MGA deletion Leads to Richter's Transformation Via NME1

MGA deletion Leads to Richter's Transformation Via NME1

Safety and Efficacy of AUTO1, a Fast-Off Rate CD19 CAR in Relapsed/Refractory B-Cell Non-Hodgkin's Lymphoma (B-NHL) and Chronic Lymphocytic Leukaemia (CLL)

INTRODUCTIONWe have previously described AUTO1, a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce CAR-T immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL. Cumulatively, this data confirms the intended function of the receptor, with low levels of CRS/ICANS and long-term engraftment of CAR T-cells observed in both patient groups. Recently, CAR-T therapy has been explored in indolent lymphomas such as follicular (FL) and mantle cell lymphoma (MCL), but a high incidence of toxicity including Grade 3-4 ICANS has been reported. We have initiated testing of AUTO1 in the setting of indolent and high-grade B-NHL and CLL (NCT02935257).METHODSManufacturing: CAR T-cell products were generated using a semi-automated closed process from non-mobilised leukapheresate.Study design: Subjects ≥ 16y underwent lymphodepletion with fludarabine (30mg/m 2 x3) and cyclophosphamide (60mg/kg x1) prior to AUTO1 infusion, with the exception of the DLBCL cohort who additionally received a single dose of pembrolizumab (200mg) on day -1 to potentiate CAR-T expansion. AUTO1 dose varies based on the indication. Split dosing of 230 x10^6 CD19 CAR T-cells at day 0 and day 9 is employed in the CLL cohort. A single dose of 200 x10^6 CD19 CAR T-cells is delivered to patients with B-NHL. Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months.RESULTSAs of 17th May 2021, we recruited 13 patients: 7 with FL, 4 with MCL, 1 DLBCL and 1 CLL. Apheresis and product manufacture was successful in all 13 patients and 9 patients were infused: 7 with FL and 2 with MCL. Three patients (1 DLBCL, 1 CLL and 1 MCL) were pending infusion at time of data cut-off and 1 patient (MCL) died due to Covid-19 prior to infusion. Patients treated with AUTO1 had a median age of 56 years (range 39-68y), had received a median of 3 prior lines of treatment (range 2-5) and all patients had stage IV disease at screening. Grade 1 CRS was reported in 4/9 and Grade 2 CRS in 1/9. 1/9 developed MAS which resolved with anakinra/dexamethasone. No ICANS was observed on study. Excellent CAR engraftment was observed and 9/9 patients were in CMR by 18FDG PET-CT post-treatment. At a median of 6.1 months (range 4.0-8.1m), 8/9 patients were disease free at last follow-up. One patient died in CMR at month 5.6 of COVID-19.CONCLUSIONAUTO1 has a tolerable safety profile in adult patients with r/r B-NHL despite high disease burden. Early data shows 100% complete remission rates and excellent CAR engraftment/expansion. Additional MCL, CLL and DLBCL patients, updated data and longer follow up will be presented. DisclosuresRoddie: Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau. Hartley: Astra Zeneca: Ended employment in the past 24 months; ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Farzaneh: Autolus: Consultancy, Current equity holder in publicly-traded company. Lowdell: Autolus: Consultancy, Current equity holder in publicly-traded company. Linch: Autolus: Consultancy, Current equity holder in publicly-traded company. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company. Peggs: Autolus: Consultancy, Current equity holder in publicly-traded company.

Statins in mature B-cell lymphomas and leukaemias.

Statins in mature B-cell lymphomas and leukaemias.

CLL-Specific B Cell Receptor Immunoglobulin Stereotypes Are Very Infrequent in Low Count Monoclonal B Cell Lymphocytosis: Implications for Disease Ontogeny

In chronic lymphocytic leukemia (CLL), the existence of subsets of patients with stereotyped B cell receptor immunoglobulin (BcR IG), collectively accounting for ~30% of all CLL, strongly implicates antigen selection in disease ontogeny. That said, it is still inconclusive if and how frequently “CLL-specific” BcR IG gene rearrangements occur in the repertoire of unaffected individuals. This is especially relevant when also considering monoclonal B-cell lymphocytosis (MBL), detected in 3-12% of the general population and characterized by the presence of CLL-like clonal B cells in lower numbers compared to CLL with no evidence of disease. MBL is distinguished into high and low count (HC-MBL and LC-MBL, respectively) with the former considered as a pre-leukemic condition, while the latter having an unclear, if any, link to CLL. Our previous immunogenetic studies have revealed similarities between HC-MBL and CLL, thus setting them apart from LC-MBL that displayed distinct IG gene usage and low incidence of stereotypy. However, these studies were Sanger-based, thus inherently limited regarding sensitivity and accurate quantification.Here we sought to overcome these limitations and obtain a comprehensive view into the composition of the IG repertoire in LC-MBL by NGS immunoprofiling, searching particularly for major CLL IG stereotypes. The study group comprised 23 individuals with CLL-like (CD5+) LC-MBL identified by a standardized flow cytometry protocol. PBMCs were the starting material in 11 cases, whereas sorted MBL cells were studied in the remaining 12 cases; in 9 cases, the normal B cell compartment was also flow sorted. Included in the analysis were also 6 individuals without LC-MBL in whom we sorted different B cell subpopulations, including naïve B cells (n=6) and memory B cells (n=5). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified with leader primers and sequencing libraries were prepared with the TruSeq DNA LT Kit and run on the MiSeq Sequencer using the MiSeq Reagent Kit v3. NGS reads were subjected to an in-house bioinformatics pipeline for clonotype characterization and the identification of CLL-specific BcR IG stereotypes.Overall, 16,857,788 productive filtered-in sequence reads were obtained corresponding to an average of 366,474 reads/sample (range: 67,175-662,994). We identified very few reads (2,470/16,857,788; 0.15%) corresponding to 17/19 major CLL BcR IG stereotypes. Their incidence in LC-MBL did not follow the CLL trend: for instance, the stereotypic IGHV3-21 gene rearrangement defining subset #2, the most frequent in CLL, accounted for only 27/2,470 reads (1.1%) corresponding to major subset stereotypes. Overall, stereotypes were identified in 26/47 samples (55%) from 13/23 LC-MBL and 6/6 non LC-MBL cases. In LC-MBL cases bearing stereotypes the breakdown according to sample type was as follows: (i) PBMCs, n=3 (ii) normal B cell compartment, n=7; (iii) sorted MBL cells, n=5: the stereotypes detected in this particular sample type were present at very low frequencies and concerned rearrangements with unmutated IGHV genes. In individuals without LC-MBL, stereotypes were detected in all 6 samples from naïve B cells versus only 2/5 samples from memory B cells. In general, we did not identify significant associations between specific B cell subpopulations and particular stereotypes, except for the IGHV4-34 rearrangement defining CLL subset #4, an indolent CLL variant with features of BcR-anergized clones, which was found only within naïve B cells.In conclusion, we demonstrate that LC-MBL is immunogenetically distinct from CLL. Major CLL BCR IG stereotypes are very scarce in individuals with or without LC-MBL. This could be taken to imply either that these stereotypes are indeed scarce in the blood of individuals unaffected by CLL or, alternatively, that they might reside within other B cell compartments not assessed in this study. DisclosuresAgathangelidis:Gilead Sciences: Research Funding. Stamatopoulos:Janssen Pharmaceuticals: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Novartis SA: Research Funding. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Research Funding.

Effects of Green Tea on Various Types of Indolent Low Grade B-Cell Lymphomas

Low grade B cell neoplasms, such as chronic lymphocytic leukemia (CLL), usually are asymptomatic when diagnosed and undergo close observation. Many patients with low grade B cell lymphomas and leukemias (LGBCL) appreciate reassurance that not treating at time of diagnosis is the appropriate management. Society has long focused on diet and lifestyle changes as an alternative to medication; however, there is limited data to which these changes can assist with hematologic disorders. Green tea (GT) has been studied in regard to its effects on longevity and health. A cohort study followed 52,000 adults over 13 years and found that the frequency of GT consumption was inversely associated with risk of total hematologic neoplasms (Takada M, et al., 2019). There are several other studies that report key oncologic pathways are disrupted by a compound found in GT called epigallocatechin-gallate (EGCG) (Farooqi A, et al., 2020). Following a case series publication by Dr. Shanafelt, the National Cancer Institute sponsored clinical trials through the Mayo Clinic, evaluating EGCG in 42 patients with asymptomatic CLL. Patients received 2000mg EGCG twice daily for 6 months. Results showed 69% of patients had a biological response with greater than 20% reduction in absolute leukocyte count (ALC) and/or greater than 30% in reduction in size of the lymph node area utilizing standard assessment criteria (Shanafelt T. et al. 2013). This data encouraged our practice to recommend to patients to drink two bags of GT in steamy hot water daily. We studied 11 patients with indolent CLL, follicular lymphoma (FL), Waldenstrom macroglobulinemia (WM), monoclonal gammopathy of undetermined significance (MGUS), and splenic Marginal zone lymphoma (MZL). No patient was symptomatic. Disease makers were recorded over time during their routine visits for observation. The markers used were ALC for CLL and MZL, standardized uptake value (SUV) on PET scan for lymph node detection for FL, immunoglobulin level as well as monoclonal protein (M-protein) measured by SPEP for MGUS, and IgM and M-protein for WM. One patient with extensive cutaneous plaque psoriasis had routine skin checks. The results were recorded in the electronic health record over varying time periods based on patient presentation and follow up. Six patients with CLL responded to GT, with reductions in ALC. In patient 1, ALC decreased by 37% over 48 months. Patient 2's ALC decreased by 68% over 102 months. Patient 3's ALC decreased by 74% in 96 months. Patient 4 had a drastic increase in ALC from 23,900 to 72,400 over three years before GT. Once starting GT, his ALC plateaued at 72,000 and has remained stable for 65 months. Patient 5 had decrease ALC of 66% over 99 months. Patient 6 with WM had a 79% decrease in IgM over 65 months drinking GT. M-protein decreased by 81%. Patient 7 had a decrease in IgM by 26% and a 24% decrease in M-protein over 36 months. Patient 8 with MGUS had a decrease in IgG by 25% over 77 months. Patient 9 had a decrease in IgG by 23% with M-protein reduction of 43%. Due to compliance issues, duration of therapy was unknown. Patient 10 with ZL had an ALC reduction of 73% over 95 months. Lastly, patient 11 with FL and psoriasis was treated with GT for 23 months. There was a reduction of external iliac and inguinal lymph node SUV by 38% and 83% over 23 months, respectively. It was also noted of a significant improvement of extensive plaque psoriasis during this time on GT (Table 1). The 11 patients highlighted in the practice had improvement in biomarkers and lymphadenopathy during the time they took daily GT. The EGCG study at Mayo Clinic, in which CLL patients took up to 4,000mg of EGCG per day, had significant responses as well as adverse effects including transaminitis. This compares to the population in our study, which includes various types of LGBCL and MGUS that were taking at most 200mg of EGCG per day and had significant responses with no adverse effects. The use of low dose EGCG for inducing clinical improvement of these types of LGBCL and MGUS may be used to delay the onset of starting therapy, thus forestalling immunosuppressive treatment and associated toxicities. Larger randomized control trials involving other forms of indolent LGBCL and MGUS would be significant for the potential of GT being a safe, non-toxic lifestyle change that could benefit patients who do not require aggressive medical therapy for their neoplastic processes. Disclosures No relevant conflicts of interest to declare.

A Phase I Study of CD19-Targeted 19(T2)28z1xx CAR T Cells in Adult Patients with Relapsed or Refractory B-Cell Malignancies

Background: Autologous CAR T cell therapy targeting the B-cell specific surface antigen CD19 has demonstrated favorable clinical responses in relapsed or refractory (R/B) B-cell lymphomas (BCL). However, despite 40-60% initial complete response (CR) rates, only a subset of patients experience durable remissions, and there is a need to further improve the efficacy of CAR therapies by preventing relapse and attaining a deeper CR. We hypothesized that the redundancy of CD28 and CD3V signaling in a CAR design incorporating all 3 CD3Vimmunoreceptor tyrosine-based activation motifs (ITAMs) might foster counterproductive T cell differentiation and exhaustion, and therefore created a new CD19 CAR construct with calibrated CAR activation potential by mutating 2 of the 3 ITAMs, termed 1XX. In systemic ALL mouse models, 19-28z1XX CAR induced effective tumor eradication at low CAR T cell doses with improved survival compared to conventional 19-28z CAR. Further preclinical studies demonstrated that the enhanced therapeutic benefit resulted from the reduced strength of activation mediated by the 19-28z1XX CAR, achieving a favorable balance of effector and memory functions, thereby enhancing persistence of functional CAR T cells and promoting effective elimination of CD19+ leukemia at lower T cell doses than needed with 19-28z CAR T cells (Feucht J et al. Nat Med 2019). To further improve the persistence of functional CAR T cells, we screened different humanized CD19-directed scFv in the context of a 19-28z1XX CAR design and proved high specificity and functionality of 19-28z1XX CARs containing a novel humanized scFv T2 - termed 19(T2)28z1XX. Study Design and Methods: This study is a single center Phase I clinical trial of 19(T2)28z1XX in patients with R/R B-cell malignancies at Memorial Sloan Kettering Cancer Center (NCT04464200). Key disease eligibility criteria include R/R diffuse large B cell lymphoma (DLBCL), high grade BCL, primary mediastinal BCL, indolent BCL and chronic lymphocytic leukemia (CLL). Patients with prior CD19 CAR therapies are eligible as long as expression of CD19 is confirmed. Key exclusion criteria include ongoing immunosuppression such as systemic GvHD therapy and active CNS disease. The study uses a 3+3 dose-escalation design to identify the maximum tolerated dose for BCL. There are 5 planned flat-dose levels. Patients will receive conditioning chemotherapy consisting of 3 days of fludarabine and cyclophosphamide followed by a single infusion of 19(T2)28z1XX CAR T cells. In the dose-escalation phase, patients with DLBCL, high grade BCL, and primary mediastinal BCL are eligible to participate. Once the recommended phase 2 dose (RP2D) is determined, the study will open to dose expansion phase with two cohorts. Cohort 1 includes DLBCL, high grade BCL and primary mediastinal BCL (i.e. same eligibility criteria as the dose-escalation phase). Cohort 2 will include patients with indolent BCL, CLL, and Richter's transformation. The dose-expansion part of the trial is designed to further characterize the safety, efficacy, and pharmacokinetics of 19(T2)28z1XX CAR in multiple indications. The primary objective of the trial is to evaluate safety and tolerability and determine the recommended Phase 2 dose of 19(T2)28z1XX. Key secondary objectives include evaluation of 19(T2)28z1XX's efficacy and cellular kinetics. Exploratory objectives include assessment of B cell aplasia, and analysis of serum cytokines. The trial has begun enrollment in August 2020. The investigators are hopeful this study will lead to development of improved CD19 CAR T cell therapy with enhanced efficacy and favorable toxicity profiles with lower infused T cell dose. Disclosures Park: AstraZeneca: Consultancy; Servier: Consultancy, Research Funding; Autolus: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy; Minverva: Consultancy; Artiva: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Kite: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Genentech/Roche: Research Funding; Juno Therapeutics: Research Funding; GSK: Consultancy; Intellia: Consultancy; Allogene: Consultancy. Riviere:Fate Therapeutics Inc.: Consultancy, Other: Ownership interest , Research Funding; FloDesign Sonics: Consultancy, Other: Ownership interest; Juno Therapeutics: Other: Ownership interest, Research Funding; Takeda: Research Funding; Atara: Research Funding. Palomba:Genentech: Research Funding; Juno Therapeutics, a Bristol-Meyers Squibb Company: Honoraria, Research Funding; Regeneron: Research Funding; Novartis: Honoraria; Merck: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria. Brentjens:BMS: Research Funding; Gracell Therapeutics: Consultancy; Juno Therapeutics (a Bristol Myers Squibb company): Patents &amp; Royalties. Sadelain:Atara: Patents &amp; Royalties, Research Funding; Fate Therapeutics: Patents &amp; Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents &amp; Royalties; Mnemo: Patents &amp; Royalties; Takeda: Patents &amp; Royalties, Research Funding. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as conditioning therapy prior to 19(T2)28z1XX CAR T cell administration.

Expression of Sf3b1-K700E accelerates the Development of Chronic Lymphocytic Leukemia in a Del(13q) Murine Model

Expression of Sf3b1-K700E accelerates the Development of Chronic Lymphocytic Leukemia in a Del(13q) Murine Model

Dysregulation of different classes of tRNA fragments in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and dysregulation of tRNA-derived short noncoding RNA (tsRNA) (tRF-1) expression is an accompanying event in the development of this disease. tsRNAs are fragments originating from the 3' end of tRNA precursors and do not contain mature tRNA sequences. In contrast to tsRNAs, mature tRFs (tRF-3s, tRF-5s, and internal tRFs) are produced from mature tRNA sequences and are redundant fragments. We investigated tsRNA expression in CLL and determined tsRNA signatures in indolent CLL and aggressive CLL vs. normal B cells. We noticed that both ts-43 and ts-44 are derived from distinct genes of pre-tRNAHis, and are down-regulated in CLL 3- to 5-fold vs. normal B cells. Thus, we investigated expression levels of tRF-5 fragments from tRNAHis in CLL samples and healthy controls, and determined that such fragments are down-regulated by 5-fold in CLLs vs. normal controls. Given these results, we investigated the expression of all mature tRFs in CLLs vs. normal controls. We found a drastic dysregulation of the expression of mature tRFs in CLL. In aggressive CLL, for the top 15 up-regulated fragments, linear fold change varied from 2,053- to 622-fold. For the top 15 down-regulated fragments in CLL, linear fold change varied from 314- to 52-fold. In addition, 964 mature tRFs were up-regulated at least 2-fold in CLL, while 701 fragments were down-regulated at least 2-fold. Similar results were obtained for indolent CLL. Our results suggest that mature tRFs may have oncogenic and/or tumor suppressor function in CLL.

Time to Second Treatment As a Proxy for Overall Survival in CLL/SLL: Identifying Risk Factors to Help Guide Treatment Selection

Time to Second Treatment As a Proxy for Overall Survival in CLL/SLL: Identifying Risk Factors to Help Guide Treatment Selection

Computed tomography textural analysis for the differentiation of chronic lymphocytic leukemia and diffuse large B cell lymphoma of Richter syndrome.

To test the hypothesis that both indolent and aggressive chronic lymphocytic leukemia (CLL) can be differentiated from diffuse large B cell lymphoma (DLBCL) of Richter syndrome (RS) by CT texture analysis (CTTA) of involved lymph nodes. We retrospectively included 52 patients with indolent CLL (26/52), aggressive CLL (8/52), and DLBCL of RS (18/52), who underwent standardized contrast-enhanced CT. In main lymphoma tissue, VOIs were generated from which CTTA features including first-, second-, and higher-order textural features were extracted. CTTA features were compared between the entire CLL group, the indolent CLL subtype, the aggressive CLL subtype, and DLBCL using a Kruskal-Wallis test. All p values were adjusted after the Bonferroni correction. ROC analyses for significant CTTA features were performed to determine cut-off values for differentiation between the groups. Compared with DLBCL of RS, CTTA of the entire CLL group showed significant differences of entropy heterogeneity (p < 0.001), mean intensity (p < 0.001), mean average (p = 0.02), and number non-uniformity gray-level dependence matrix (NGLDM) (p = 0.03). Indolent CLL significantly differed for entropy (p < 0.001), uniformity of heterogeneity (p = 0.02), mean intensity (p < 0.001), and mean average (p = 0.01). Aggressive CLL showed significant differences in mean intensity (p = 0.04). For differentiation between CLL and DLBCL of RS, cut-off values for mean intensity and entropy of heterogeneity were defined (e.g., 6.63 for entropy heterogeneity [aggressive CLL vs. DLBCL]; sensitivity 0.78; specificity 0.63). CTTA features of ultrastructure and vascularization significantly differ in CLL compared with that in DLBCL of Richter syndrome, allowing complementary to visual features for noninvasive differentiation by contrast-enhanced CT. • Richter transformation of CLL into DLBCL results in structural changes in lymph node architecture and vascularization that can be detected by CTTA. • First-order CT textural features including intensity and heterogeneity significantly differ between both indolent CLL and aggressive CLL and DLBCL of Richter syndrome. • CT texture analysis allows for noninvasive detection of Richter syndrome which is of prognostic value.

Role of long non-coding RNAs in disease progression of early stage unmutated chronic lymphocytic leukemia.

Predicting disease progression in chronic lymphocytic leukemia (CLL) remains challenging particularly in patients with Rai Stage 0/I disease that have an unmutated immunoglobulin heavy chain variable region (UM IGHV). Even though patients with UM IGHV have a poor prognosis and generally require earlier treatment, not all UM IGHV patients experience more rapid disease progression with some remaining treatment free for many years. This observation suggests biologic characteristics other than known prognostic factors influence disease progression. Alterations in long non-coding RNA (lncRNA) expression levels have been implicated in diagnosis and prognosis of various cancers, however, their role in disease progression of early Rai stage UM CLL is unknown. Here we use microarray analysis to compare lncRNA and mRNA profiles of Rai 0/I UM IGHV patients who progressed in <2 years relative to patients who had not progressed for >5 years. Over 1,300 lncRNAs and 940 mRNAs were differentially expressed (fold change ≥ 2.0; p-value ≤ 0.05). Of interest, the differentially expressed lncRNAs T204050, NR_002947, and uc.436+, have known associated genes that have been linked to CLL. Thus, our study reveals differentially expressed lncRNAs in progressive early stage CLL requiring therapy versus indolent early Rai stage UM CLL. These lncRNAs have the potential to impact relevant biological processes and pathways that influence clinical outcome in CLL.