Untreated Chronic Lymphocytic Leukemia Patients Research Articles

Abstract 5842: An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia

Abstract Therapeutic inhibition of BCR signaling using Bruton tyrosine kinase inhibitors (BTKis) has remarkable efficacy in the treatment of chronic lymphocytic leukemia (CLL). However, the efficacy of BTKis is limited by the development of drug resistance, which can be mediated through mutations in genes encoding proteins of BCR signaling pathways. Approaches targeting the BCR mutant proteins have been limited by the large heterogeneity of these mutations. In addition, BCR signaling mutations are only detected in up to two-thirds of the CLL patients who progressed with BTKi treatment, suggesting mechanisms beyond genetic mutations. We previously showed that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells (Wang et al., Blood Cancer J. 2022), suggesting that epigenetic mechanisms are exploited by CLL B cells to support their survival. To gain insights into the epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome-wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on BTKi ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in a CLL cohort at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies showed that suppressing BCR signaling by BTKi leads to the downregulation of genes associated with mitochondrial translation, a key process of mitochondrial biogenesis. We also found decreased chromatin accessibility and H3K27ac deposition at the promoters of these MT genes. Interestingly, in patients who experienced disease progression on ibrutinib, there was a restoration of MT gene expression, chromatin accessibility, and H3K27ac levels at the MT gene promoters similar to baseline samples, suggesting an epigenetic-regulated MT both in BTKi treatment naïve and relapsed CLL patients. Importantly, MT inhibition led to a decreased survival of primary CLL B cells. These findings point toward an epigenetic mechanism utilized by BCR signaling to promote MT and leukemic cell survival. We then observed that the transcription factor NRF1 is enriched at the MT promoter in CLL B cells. CRISPR-mediated depletion of NRF1 led to reduced mitochondrial translation and cell survival in BCR signaling active malignant B cell lines and primary CLL B cells. These results suggest that NRF1 controls an epigenetic program linking BCR signaling to control MT, which in turn supports BCR signaling-dependent CLL B cell survival. Our findings demonstrated that BCR signaling utilizes epigenetic mechanisms that regulate mitochondrial metabolism to enhance cell survival in both treatment naïve and BTKi-relapsed CLL. These findings may provide new therapeutic directions for untreated and BTKi-relapsed CLL patients. Citation Format: Olivia M. Depies, Sutapa Sinha, Sameer Parikh, Neil Kay, Zhiquan Wang. An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5842.

Final results on effectiveness and safety of Ibrutinib in patients with chronic lymphocytic leukemia from the non-interventional FIRE study.

We conducted an observational study (FIRE) to understand the effectiveness and safety outcomes of ibrutinib in patients with chronic lymphocytic leukemia (CLL) in France, after a maximum follow-up of five years. Patients were included according to the French marketing authorization in 2016 (i.e. patients with relapsed or refractory CLL or to previously untreated CLL patients with deletion 17p and/or tumor protein p53 mutations unsuitable for chemoimmunotherapy) and could have initiated ibrutinib more than 30days prior their enrolment in the study (i.e. retrospective patients) or between 30days before and 14days after their enrolment (i.e. prospective patients). The results showed that in the effectiveness population (N = 388), the median progression-free survival (PFS) was 53.1 (95% CI: 44.5-60.5) months for retrospective patients and 52.9 (95% CI: 40.3-60.6) months for prospective patients and no difference was shown between the PFS of patients who had at least one dose reduction versus the PFS of patients without dose reduction (p = 0.7971 for retrospective and p = 0.3163 for prospective patients). For both retrospective and prospective patients, the median overall survival was not reached. The most frequent treatment-emergent adverse event of interest was infections (57.6% retrospective; 71.4% prospective). A total of 14.6% of the retrospective patients and 22.4% of the prospective patients had an adverse event leading to death. Our findings on effectiveness were consistent with other studies and the fact that patients with dose reductions had similar PFS than patients without dose reduction is reassuring. No additional safety concerns than those already mentioned in previous studies could be noticed.Trial registrationClinicalTrials.gov, NCT03425591. Registered 1 February 2018 - Retrospectively registered.

SOLUBLE CD150 ISOFORM LEVEL IN PLASMA OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS.

SLAMF1/CD150 is an active player in B cell signaling networks in chronic lymphocytic leukemia (CLL). CD150-mediated signaling initiates through a homophilic CD150 binding, which spans the adjacent cells, or the interaction with the soluble CD150 isoform (sCD150). The expression of sCD150 isoform at the mRNA and protein levels ex vivo was confirmed. However, it is unclear whether sCD150 isoform present in the blood plasma of CLL patients is a factor in the constitutive activation of CD150+ cells. The aim of this study was to develop an ELISA assay for the specific sCD150 evaluation and assess the sCD150 levels in the blood plasma of CLL patients with different CD150 expression on B cells. Blood plasma samples and peripheral blood mononuclear cells from 40 previously untreated CLL patients were analyzed. An ELISA method, ex vivo drug sensitivity assay, and a cell viability assay were used. The sCD150 isoform was found in all studied plasma samples of CLL patients at different levels regardless of the cell surface CD150 expression status of B cells and sCD150 mRNA expression. CLL cases with low levels of the cell surface CD150 expression in B cells are characterized by high levels of sCD150 in blood plasma in contrast to the CLL cases with high cell surface CD150 expression on B cells. The elevated levels of sCD150 in blood plasma are associated with a better sensitivity of malignant B cells to cyclophosphamide and bendamustine. The sCD150 isoform is actively secreted by CLL B cells with its accumulation in blood plasma, which may be regarded as an additional factor in the CLL clinicopathologic variability.

Prognostic value of clonal evolution identified by sequential FISH in untreated chronic lymphocytic leukaemia

Aim: The aim of the current study was to evaluate the potential clinical impact of clonal evolution detected by fluorescence in situ hybridization (FISH) in untreated chronic lymphocytic leukaemia (CLL) patients managed with a watch-and-wait strategy. Methods: We performed both overall survival (OS) and time to first treatment (TTFT) analysis. For the first one, we exploited a real-life cohort of 123 consecutive CLL patients followed at our institution, for which at least a second FISH evaluation during watch and wait was available. For TTFT analysis, we considered only patients treated after the second FISH sample (n = 69). Results: Considering the original cohort, patients who acquired a FISH abnormality displayed a worse outcome with a median OS of 91.9 months compared to 147.3 months for patients who did not acquire any FISH abnormalities (P = 0.007). Unmutated immunoglobulin heavy chain gene (IGHV) genes were associated with a higher probability of acquiring a FISH abnormality (P = 0.04). Turning to TTFT analysis, patients who gained at least one FISH abnormality (n = 7, 10%) were characterised by an earlier treatment requirement with a median TTFT of 1.1 months, compared to 2.7 months in patients who did not acquire any FISH abnormalities (n = 62, 90%) (P = 0.025). Conclusions: The dynamic acquisition of karyotypic abnormalities by FISH predicts poor outcomes and early treatment requirement in CLL patients. Our results suggest that FISH analysis could be integrated with other clinical and biological features to obtain dynamic scores that are able to predict outcomes at different phases of disease history.

IMMUNOPHENOTYPE OF LEUKEMIC CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH NOTCH1 AND SF3B1 GENE MUTATIONS.

The typical chronic lymphocytic leukemia (CLL) immunophenotype is vital for diagnosis, but the expression of some antigens varies and has prognostic value. There are data that reduced CD20 expression is associated with NOTCH1 and SF3B1 gene mutations. To determine a high-risk group of CLL patients for prediction of unfavorable NOTCH1 and SF3B1 gene mutations based on immunophenotyping of leukemic cells. Flow cytometric and molecular-genetic analysis (mutations of NOTCH1, SF3B1, and TP53 genes using the polymerase chain reaction followed by direct sequencing) was performed in a group of 86 previously untreated CLL patients. The immunophenotype of leukemic cells of all examined patients met the criteria of CLL diagnosis. NOTCH1 gene mutations were found in 21 patients (24.4%), and SF3B1 gene mutations - in 7 patients (8.1%). There were no TP53 gene mutations among the examined patients. A decreased number of CD20+CD5+ cells and a downward trend in the relative index of mean fluorescence intensity (iMFI) of CD20+ cells were found in patients with NOTCH1 and SF3B1 gene mutations. Based on the iMFI level (higher and/or lower than 3.0) and the number of CD20+CD5+ cells among all B-cells (higher and/or lower than 50%), we distinguished CLL cases with low and relatively high levels of CD20 antigen expression. Using ROC analysis and the parameter of low CD20 antigen expression, we could predict the presence of NOTCH1 and SF3B1 gene mutations in 73.3 ± 0.06% of patients (p = 0.001). The risk of NOTCH1 and SF3B1 gene mutations in cases with low CD20 antigen expression was 6.96 (95% CI = 2.53-19.18; p = 0.0001). The revealed regularities were statistically significant for patients in whom the diagnosis was established in all Binet - Rai stages except A0-AI. Our data confirmed a reduced CD20 expression in CLL patients with NOTCH1 and SF3B1 mutations. In addition, an approach was proposed to identify high-risk CLL patients for prediction of such mutations: previously untreated CLL patients at advanced Binet - Rai stages (BII, CIII, CIV) with a reduced number of double-positive CD20+CD5+ cells in peripheral blood and/or low iMFI of CD20+ cells.

Integrative NGS testing reveals clonal dynamics of adverse genomic defects contributing to a natural progression in treatment-naïve CLL patients.

Large-scale next-generation sequencing (NGS) studies revealed extensive genetic heterogeneity, driving a highly variable clinical course of chronic lymphocytic leukaemia (CLL). The evolution of subclonal populations contributes to diverse therapy responses and disease refractoriness. Besides, the dynamics and impact of subpopulations before therapy initiation are not well understood. We examined changes in genomic defects in serial samples of 100 untreated CLL patients, spanning from indolent to aggressive disease. A comprehensive NGS panel LYNX, which provides targeted mutational analysis and genome-wide chromosomal defect assessment, was employed. We observed dynamic changes in the composition and/or proportion of genomic aberrations in most patients (62%). Clonal evolution of gene variants prevailed over the chromosomal alterations. Unsupervised clustering based on aberration dynamics revealed four groups of patients with different clinical behaviour. An adverse cluster was associated with fast progression and early therapy need, characterized by the expansion of TP53 defects, ATM mutations, and 18p- alongside dynamic SF3B1 mutations. Our results show that clonal evolution is active even without therapy pressure and that repeated genetic testing can be clinically relevant during long-term patient monitoring. Moreover, integrative NGS testing contributes to the consolidated evaluation of results and accurate assessment of individual patient prognosis.

Transcriptomic Signature of a Patient with Chronic Lymphocytic Leukemia Exhibiting Profound Fluctuations in the Lymphocytosis during Nutrition and Prolonged Fasting Periods

Introduction This study aimed to investigate the underlying reasons behind sustained significant continuous deep fluctuations in the absolute lymphocyte count (ALC) in an untreated patient with Chronic Lymphocytic Leukemia (CLL), who has maintained a favorable prognosis since the time of diagnosis in 2016. The patient has voluntarily chosen to adopt a mainly vegetarian and fruitarian diet, including annual periods of prolonged total fasting (lasting from 4 to 39 days). Methods For this purpose, we decided to conduct a comprehensive analysis of the whole transcriptome profiling of peripheral blood (PB) CD19+ cells from patient (#1) at different time-points, in comparison to the corresponding cells from 5 other untreated CLL patients who followed a varied diet. As a result, the CLL patients were classified as follows: the 1st group consisted of patient #1 at 20 different time-points (16 time-points during nutrition and 4 time-points during fasting), whereas the 2nd group included only one time point for each of the patients (#2, #3, #4, #5, and #6) due to their varied dietary practices. We have performed microarray experiments on the Peripheral Bone Marrow Cells (PBMCs) CD19+ cells utilizing the Affymetrix Human Clariom™ D Pico Assay, a powerful tool for generating high-fidelity biomarker signatures. The data was preprocessed and normalized using the Robust Multiarray Average (RMA) method. Significance Analysis of Microarrays (SAM version 3.0) was utilized to assess significant differentially expressed genes between the 1st group and 2nd group of CLL patients. The Storey and Tibshirani procedure was applied to control the False Discovery Rate (FDR). Results The lymphocytosis trend of patient #1 displayed recurring significant deep fluctuations since the time of diagnosis. Interestingly, we observed that approximately 4-6 weeks after the conclusion of fasting periods, the absolute lymphocyte count was reduced by about half (Figure 1A). On the other hand, in the 2nd group, all patients did not show fluctuations in the absolute lymphocyte count, but instead demonstrated a progressive increase in lymphocytes over time. Differential expression analysis, after correction for multiple testing, identified 18 differentially expressed genes (DEGs) in the comparison between CD19+ cells of patient #1 and patients #2, #3, #4, #5, and #6. Among them, our focus was on 9 genes with functional annotation: 4 genes were downregulated ( IGLC3, RPS26, CHPT1, and PCDH9), and 5 genes were upregulated ( IGHV3-43, IGKV3D-20, PLEKHA1, CYBB, and GABRB2) in the 1st group (patient #1) vs. the 2nd group of patients (#2, #3, #4, #5, and #6), respectively. In particular, IGLC3, RPS26, PLEKHA1, CYBB, and GABRB2 showed high Fold Change (FC) > 3. Additionally, we generated a heat map on log2 expression of the 9 differentially expressed genes across different patients/conditions (Figure 1B). Furthermore, hierarchical clustering analysis revealed that patient #1 during nutrition and fasting (1st group) clustered distinctly from all CLL patients (2nd group), indicating a gene expression signature that differentiated patient #1 from patient #2, #3, #4, #5, and #6. Conclusions In conclusion, our transcriptomic study revealed a small set of 9 genes that distinguished an untreated CLL patient who underwent a prolonged periods of total fasting and maintained a slow-growing tendency of lymphocytosis, in comparison to 5 untreated CLL patients with a varied diet. Future investigations of patient #1 could demonstrate the potential role of prolonged periodic fasting and the involvement of the 9 genes in sustaining the trend of lymphocytosis and the benign course of the disease.

Higher Mutational Burden Is an Independent Predictor of Shorter Time to First Treatment in Untreated Chronic Lymphocytic Leukemia Patients

Background. Recent advancements in next-generation sequencing (NGS) technologies have revolutionized the ability to comprehensively profile chronic lymphocytic leukemia (CLL), unveiling various molecular lesions. Complex mutational landscapes and diverse combinatorial patterns of genetic alterations lead to heterogeneity of clinical course and outcomes. The primary objective of this study was to identify patterns of co-occurrence (CO) and mutual exclusivity (ME) among mutations in CLL and their impact on time to first treatment (TTFT). Methods. The cohort included 795 treatment-naïve (TN) patients (pts) with CLL diagnosed according to iwCLL guidelines. The median follow-up time for the cohort was 3.91 years (range 0 - 42.8). The median time from diagnosis to mutational analysis was 6.6 months (26.50 - 33.27, 95% CI). Mutational screening was performed by a clinical targeted NGS panel, covering all or hotspot exons of 95 genes - the Rapid Heme Panel implemented by BWH (Kluk MJ, et al. J Mol Diagn. 2016;18(4):507-515). Standard protocols were applied to analyze IGHV gene mutational status, chromosomal aberrations by FISH with probes for 11q, 13q, 17p, and trisomy 12, and stimulated karyotype, where complex karyotype (CK) was defined as ≥3 abnormalities. CO and ME patterns were analyzed in pts with a higher mutational burden, determined as ≥2 distinct mutated genes, using Fisher's exact test ( p<.05). Control for false discovery rate was performed using the Benjamini, Krieger and Yekutieli method ( q=20%). TTFT was the time from diagnosis to initial treatment. Results. We detected 1798 mutations in 719 (90.4%) pts. No mutations were detected in 9.6% (n=76) patients, while a single mutated gene was found in 216 (27.2%) pts, 2 mutated genes in 206 (25.9%) pts, and mutations in 3 and ≥4 genes were found in 153 (19.2%) and 144 (18.1%) pts, respectively. The most mutated (≥10%) genes in the cohort were ATM (18.1%), NOTCH1 (17.7%), TP53 (13.2%), SF3B1 (12.3%), TET2 (10.2%), and DNMT3A (10.1%). A higher mutational burden was associated with clinical Rai stages 1-4 [ p=.003]; elevated (>3.5 mg/L) levels of baseline ß-2 microglobulin (B2M) [ p=.036] and established high-risk genomic features: del(17p) [ p<.001], del(11q) [ p=.021], CK [ p=.009], unfavorable ZAP70 [ p=.027] and unmutated IGHV (UM- IGHV) [ p<.001] (incidence of UM- IGHV cases is 48.4% (n=359) in cohort). At the same time, cases with 0-1 mutation were more likely to have del(13q) [ p=.006]. Sex and age >65 years at diagnosis were not associated with mutational burden. Otherwise, lack of any mutations was associated with absence of del(17p) (0% vs 11.1% [ p=.002]), lower rates of B2M >3.5 mg/L (3.9% vs 21.7% [ p<.001]), less frequent UM- IGHV (25.4% vs 50.9% [p<.001]), lower ratio of CK (7.0% vs 17.5% [p=.024] and age >65 years at diagnosis (21.1% vs 35.1% [ p=.014]). After controlling for multiple comparisons, no genes showed significant mutational co-occurrence. UM- IGHV cases were enriched in mutated ATM [ p=.003], NOTCH1 [ p<.00001], SF3B1 [ p=.0012] and XPO1 [ p<.00001]. Mutated NOTCH1 [ p<.00001], as well as KRAS [ p=.0012], occurred along with tri12. In contrast, UM- IGHV did not co-occur with mutations in MYD88 [ p<.00001], as previously reported. Del(13q) did not co-occur with mutated BRAF [ p=.0006] or BCOR [ p=.006]. Mutated TP53 [ p=.008] and SF3B1 [ p=.0013] were absent in tri12 positive cases. Increased mutational burden in ≥2 distinct mutated genes predicted a shorter TTFT than 0 or 1 mutated gene (HR 2.16; 95%CI 1.57 - 2.97; p<.001). Factors including Rai stages 1-4 (HR 2.63; 95%CI 1.88 - 3.7; p<0.001), UM- IGHV (HR 2.4; 95%CI 1.74 - 3.3; p<.001), CK (HR 1.47; 95%CI 1.07 - 2.0; p=.018), mutational burden in ≥2 genes (HR 1.57; 95%CI 1.14 - 2.2; p=.006) and del(17p) /TP53mut (HR 1.79; 95%CI 1.15 - 2.8; p=.01) were independently associated with worsened TTFT ( Figure 1). Neither mutated TP53 without del(17p) (HR 1.47; 95%CI 0.92 - 2.3; p=.104), or wild-type TP53 with del(17p) (HR 1.03; 95%CI 0.51 - 2.1; p=.932) were significant predictors of TTFT. Conclusions: Higher mutational burden is associated with worsened TTFT in CLL pts independently of other genomic events. In this cohort we did not see co-occurrence of specific mutations.

NK Cell Dysfunction in CLL Is Mediated through SHP-1 Signaling and Is Associated with Poor Prognosis

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, presenting with a highly variable clinical course, outcome and response to therapy. This variability has been linked to a complex tumor microenvironment and genetic/epigenetic modifications that lead to immunosuppression. Indeed, the clinical hallmark of this disease is the susceptibility to infections and secondary malignancies. While T cell dysfunction in CLL has been well described, conflicting information exists on NK cell changes in CLL and their role in CLL immunosurveillance. Here, we analyzed samples from 75 untreated (or completed treatment >2 years ago) CLL patients and 30 healthy controls. We discovered a substantial variation in NK cell function compared with healthy controls, which we further classified into hyperfunctional, normofunctional and dysfunctional based on their ability to degranulate, and produce IFN- γ in response to K562 targets ( Figure 1). NK cells from ‘dysfunctional’ CLL samples also displayed impaired cytotoxicity against K562 targets, weaker synapse formation and Syk/Zap70 signaling in response to antibody-dependent cellular cytotoxicity (ADCC) compared to those from ‘functional’ samples. The NK functional status was strongly associated with multiple prognostic markers, including cytogenetics, IgVH mutation status, ZAP-70 expression and disease stage, with patients with dysfunctional NK cells having significantly higher rates of secondary malignancies and viral infections ( Table 1). Using multispectral flow cytometry we discovered high phenotypic heterogeneity with unique NK cell clusters present in the dysfunctional samples. Those clusters express a pattern of activation-induced exhaustion, with upregulation of multiple checkpoint molecules such as PD-1, TIGIT, LAG-3, KLRG-1, TIM-3 but not evidence of terminal differentiation (CD57 negative). Those clusters also showed concomitant expression of activating molecules such as NKG2D and DNAM-1, suggesting reversibility of their dysfunctional status. We subsequently found significantly higher (p<0.0001) constitutive phosphorylation of SHP-1 protein, a phosphatase downstream of multiple inhibitory NK cell receptors, in NK cells from dysfunctional samples. Treatment with a SHP-1 inhibitor NSC-87877 (EMD Millipore) or use of siRNA targeting SHP-1 (ThermoFisher scientific), resulted in reversal of NK dysfunction with full restoration of their functional capacity. Taken together, our data suggest that NK cells functional status plays an important role in CLL disease progression and secondary complications. The novel mechanism of NK cell immune dysfunction in CLL described here has the potential to improve outcomes with novel immunotherapies such as chimeric antigen receptors, checkpoint inhibitors and bi-specific antibody therapy that have shown disappointing results in CLL to date when compared with other B cell malignancies.

Impaired T-Cell Proliferation in Chronic Lymphocytic Leukemia Patients Is Linked to Disturbed Lipid Metabolism and Homeostasis

Background Successful T-cell based anti-cancer therapies require proliferation and longevity of effector T cells, features that highly depend on metabolic processes. T cells from chronic lymphocytic leukemia (CLL) patients are characterized by acquired T-cell dysfunction, resulting in impaired activation, proliferation and cytotoxicity upon in-vitro activation (van Bruggen et al, Blood 2019). Whilst the role of glucose metabolism in activated T-cells is extensively studied, the contribution of other energy sources is less clear. Lipid metabolism has only recently come into focus as an important source of energy for immune cells. Aims Molecular and functional analysis of the lipid metabolic pathway in T cells from CLL patients. Methods Peripheral blood mononuclear cells (PBMCs) of untreated CLL patients and, as controls, age-matched healthy donors (HD) were studied. T cells at baseline and after TCR stimulation were analyzed molecularly using transcriptomics and lipidomics, and extensively characterized at protein level by flow cytometry and confocal imaging. Lipid metabolism was manipulated through culturing cells in lipoprotein deficient serum (LPDS), or in the presence of agonists and inhibitors of specific regulatory pathways. Results We found the uptake of lipids, specifically cholesterol, during T-cell activation essential for proliferation, which was evidenced by a nearly absent proliferative capacity of HD T cells in the lack of exogenous lipids which was fully rescued by re-addition of cholesterol-rich low-density lipoproteins (LDL). As CLL T cells portray an altered proliferation pattern even when cultured in normal medium, transcriptomic analysis was performed to investigate the expression of key lipid metabolism genes. CLL T-cells had reduced expression of the master transcription factors SREBP1/2, governing lipid and cholesterol metabolism, when stimulated in the presence of their autologous CLL cells (Figure 1A). Accordingly, downstream protein involved in the synthesis and desaturation of lipids and LDL uptake (specifically FASN, LDL-R and SCD) were significantly reduced in CLL compared to HD T cells. Remarkably, despite reduced capacity for LDL uptake, an accumulation of neutral lipids was found in CLL T cells especially upon stimulation (Figure 1B), a feature earlier described in dysfunctional macrophages. The lipid accumulation observed in CLL T cells was supported by the lack of expression of ATGL, a crucial enzyme for the utilization of neutral lipids for energy production and building of new membranes. In line with this, mitochondrial lipid oxidation was decreased in stimulated CLL T cells compared to HD. To investigate the composition of the accumulated lipids, lipidomics analysis was performed, revealing differential abundance of specific lipid classes in stimulated CLL versus HD T cells. Triglycerides were increased while cholesterol esters and phospholipids were reduced in CLL T cells. Triglycerides likely constitute the neutral lipids accumulation found in these cells. As cholesterol esters and phospholipids are essential for proper membrane formation, reduced levels of these species can contribute to the reduced proliferative capacity of CLL T cells. Summary/conclusion Altogether, the results presented here demonstrate a disturbed lipid composition and utilization in CLL T-cells, which contributes to T-cell dysfunction on at least two levels: decreased bioenergetics and decreased proliferation. Likewise, lipid deprivation due to both decreased uptake and a misbalanced lipidome leads to altered T-cell function. Therefore, strategies to increase lipid uptake, synthesis and utilization in order to rebalance homeostasis could ameliorate CLL T-cell dysfunction and specifically proliferation-defects in CLL.

Time-Limited Bendamustine, Rituximab, and Venetoclax (BR-VR) in Untreated Chronic Lymphocytic Leukemia (CLL): High Rates of Undetectable Minimal Residual Disease Remissions

Background: Despite the efficacy of venetoclax (VEN) in frontline CLL, optimal combination regimens and duration of treatment remain unclear. We hypothesized that cytoreduction with bendamustine/rituximab (BR) induction followed by venetoclax/rituximab (VR) consolidation for a fixed 1-year duration would be associated with an increased rate of undetectable minimal residual disease (uMRD) compared to historical controls and a reduction in the risk of tumor lysis syndrome (TLS). Here we report updated data from a fully-enrolled ongoing phase 2 multicenter, US, single-arm, open-label study (NCT03609593) designed to assess the safety and efficacy of BR-VR in previously untreated CLL patients (pts). Methods: Previously untreated CLL/SLL pts ≥ 18 years requiring therapy per iwCLL criteria initially received 3 cycles of bendamustine 50-90 mg/m 2 daily for 2 days and rituximab 375 mg/m 2 every 28 days for 3 cycles. Following BR, VEN was initiated with a standard dose escalation from 20 mg to 400 mg daily over 5 weeks. This was followed by 6 cycles of VR with rituximab given monthly and 5 cycles of VEN alone (12 cycles of VEN in total). Additional eligibility included: ECOG PS ≤ 2, hemoglobin ≥8g/dL, ANC ≥1000/mm 3, and platelets ≥50,000/mm 3. Response was assessed by 2018 iwCLL criteria with uMRD testing by central flow cytometry at a level of <10 -4 in peripheral blood (PB) and bone marrow (BM). The primary endpoint was objective response rate (ORR). Secondary endpoints included uMRD rate, time to uMRD, and adverse events (AEs) assessed by CTCAE v 5.0. Results: As of data cutoff on 15 July 2023, 42 pts were accrued. Baseline demographics were as follows: male/female (29/13), median age 61.5 yrs (range 38-84). Baseline prognostic studies showed unmutated IGHV in 22 (52%) pts, TP53 aberrant (either del(17p) and/or TP53 mutation) in 2 (5%) pt, del(11q) in 3 (7%) pts, and complex karyotype in 11 (26%) pts. TLS risk among 24 evaluable pts at baseline was high (H) in 9 (21%), medium (M) in 22 (52%), and low (L) in 6 (26%). At a median follow-up of 16.6 mo. (range, 3-40), 37 pts remain on study. Of 29 pts with at least 15 mo. follow-up (completing all therapy), the ORR was 100% (79% CR/CRi, 21% PR [due to small residual nodes]). 3 pts died on study (2 due to COVID-19 and 1 developed newly metastatic squamous cell carcinoma and taken off study after achieving a CR post-VEN ramp-up). 1 pt was lost to follow-up while in a CR. Bendamustine was administered at doses of 50 mg/m 2 in 11%, 70 mg/m 2 in 13%, and 90mg/m 2 in 76% of pts. In 38 evaluable pts, response assessments after cytoreduction with BR demonstrated 16% of pts achieved CR/CRi and 84% achieved PR. For evaluable pts at 16 mo., uMRD (<0.01%) in the PB and BM was observed in 80% (20/25) and 78% (18/23) of pts, respectively. MRD was intermediate (0.01% - <1.0%) in 17% (4 patients) in BM (Figure 1 ORR and MRD). Median time to uMRD was 11 mo. (range 3-15) in PB and 10 mo. (range 2.6-15) in BM. The most common treatment-emergent AEs during BR induction were (any grade/grade ≥3) anemia in 15/4 (36%/10%) pts, nausea in 9/0 (21%/0%), neutropenia in 12/8 (29%/19%), rash in 12/1 (29%/2%), constipation 7/0 (17%/0%), and infusion reactions in 7/1 (17%/2%). 2 pts (5%) developed febrile neutropenia during BR. Emergent AEs during VEN treatment included neutropenia in 21/13 (50%/31%), diarrhea in 18/0 (43%/0%) pts, leukopenia in 14/4 (24%/10%), and nausea in 11/0 (26%/0%). TLS risk was substantially reduced after BR lead-in. Of 9 H-risk pts at baseline, 1 remained H-risk after BR; of 16 M-risk pts, 5 remained M-risk, with the remainder at L-risk (89% reduction in H-risk TLS). Conclusions: BR-VR is a safe and well-tolerated regimen in untreated CLL pts. BR debulking substantially reduces TLS risk, and this sequential strategy achieves high rates of PB and BM uMRD across all prognostic risk groups.

LncRNA CRNDE is downregulated and associated with poor prognostic markers in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by a very heterogeneous clinical outcome. Thus, a plethora of prognostic factors and systems has been identified to place patients into different risk categories and to guide therapy decisions. The classic clinical staging models by Rai and Binet have been the cornerstone of patient management for several years. The greater insight into the molecular biology of CLL facilitated the advent of prognostic genetic biomarkers that are expected to impact clinical practice soon in the future. Therefore, we aimed to investigate the expression of long non-coding RNA (lncRNA) CRNDE in patients with CLL, and to analyze its relationship with the clinicopathological parameters of CLL. In this study, 40 untreated CLL patients and 30 age- and gender-matched controls were enrolled. The analysis of lncRNA CRNDE expression was determined using reverse transcription-quantitative polymerase chain reaction technique. Our result confirmed the downregulated expression of LncRNA CRNDE in CLL patients compared to controls (p < 0.001). The low expression of CRNDE was significantly associated with poor prognostic markers including advanced stage of CLL, high levels of serum beta-2 microglobulin and lactic dehydrogenase, and the presence of del17p (p = 0.029, p = 0.013, p = 0.003, p = 0.028; respectively). Our study demonstrated that LncRNA CRNDE is significantly downregulated and associated with poor prognostic markers in CLL. It provides a rationale to assess its biological and prognostic impact in CLL.

A Nine-Gene Expression Signature Distinguished a Patient with Chronic Lymphocytic Leukemia Who Underwent Prolonged Periodic Fasting.

Background and Objectives: This study aimed to investigate the causes of continuous deep fluctuations in the absolute lymphocyte count (ALC) in an untreated patient with Chronic Lymphocytic Leukemia (CLL), who has had a favorable prognosis since the time of diagnosis. Up until now, the patient has voluntarily chosen to adopt a predominantly vegetarian and fruitarian diet, along with prolonged periods of total fasting (ranging from 4 to 39 days) each year. Materials and Methods: For this purpose, we decided to analyze the whole transcriptome profiling of peripheral blood (PB) CD19+ cells from the patient (#1) at different time-points vs. the same cells of five other untreated CLL patients who followed a varied diet. Consequently, the CLL patients were categorized as follows: the 1st group comprised patient #1 at 20 different time-points (16 time-points during nutrition and 4 time-points during fasting), whereas the 2nd group included only one time point for each of the patients (#2, #3, #4, #5, and #6) as they followed a varied diet. We performed microarray experiments using a powerful tool, the Affymetrix Human Clariom™ D Pico Assay, to generate high-fidelity biomarker signatures. Statistical analysis was employed to identify differentially expressed genes and to perform sample clustering. Results: The lymphocytosis trend in patient #1 showed recurring fluctuations since the time of diagnosis. Interestingly, we observed that approximately 4-6 weeks after the conclusion of fasting periods, the absolute lymphocyte count was reduced by about half. The gene expression profiling analysis revealed that nine genes were statistically differently expressed between the 1st group and the 2nd group. Specifically, IGLC3, RPS26, CHPT1, and PCDH9 were under expressed in the 1st group compared to the 2nd group of CLL patients. Conversely, IGHV3-43, IGKV3D-20, PLEKHA1, CYBB, and GABRB2 were over-expressed in the 1st group when compared to the 2nd group of CLL patients. Furthermore, clustering analysis validated that all the samples from patient #1 clustered together, showing clear separation from the samples of the other CLL patients. Conclusions: This study unveiled a small gene expression signature consisting of nine genes that distinguished an untreated CLL patient who followed prolonged periods of total fasting, maintaining a gradual growth trend of lymphocytosis, compared to five untreated CLL patients with a varied diet. Future investigations focusing on patient #1 could potentially shed light on the role of prolonged periodic fasting and the implication of this specific gene signature in sustaining the lymphocytosis trend and the favorable course of the disease.

NOTCH PATHWAY MUTATION CONTRIBUTES TO INFERIOR PROGNOSIS IN HBV‐INFECTED CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) patients with hepatitis B virus (HBV) infection have a poor prognosis, underlying mechanism remains unclear. NOTCH mutations are frequent in CLL and associated with disease progression and drug resistance. It is also reported to be associated with hepatitis infection in lymphoid malignancies. In order to investigate the relation between NOTCH pathway and HBV-associated CLL, we studied 98 previously untreated HBV positive CLL patients and 244 HBV-negative CLL. A total of 342 treatment naïve patients diagnosed with CLL at the First Affiliated Hospital of Nanjing Medical University from 1 January 2010 to 31 October 2021 were enrolled in our study. The mutation hotspots of NOTCH pathway genes were analyzed by Next Generation Sequencing (NGS) of DNA that used PCR for the identification of genomic mutation. Fisher exact and χ2 tests were used to assess the correlation between HBV infection and clinical, demographic factors in CLL patients. Survival curves were generated by Graphpad 9.5. NOTCH mutations were more frequent in HBV positive CLL subgroup (17.3% vs. 7.4%, p = 0.033). By survival analysis, HBV infection was associated with disease progression and poor survival (p = 0.0099 for overall survival [OS] and p = 0.0446 for time-to-treatment [TTT]). Any lesions of the NOTCH pathway (NOTCH1, NOTCH2 and SPEN) aggravated prognosis. In multivariate analysis, NOTCH mutation retained an independent significance for HBV-infected patients (p = 0.016 for OS and p = 0.023 for TTT). However, HBV positive with NOTCH unmutated had no statistical difference in prognosis compared with HBV negative patients (p = 0.1706 for OS and p = 0.2387 for TTT), which indicated that NOTCH pathway mutation contributed to inferior prognosis in HBV-infected CLL. In conclusion, a cohort of CLL patients with HBV positive displayed a worse clinical outcome and the status of NOTCH signaling pathway might play a crucial role. Keywords: chronic lymphocytic leukemia (CLL), diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

New insight into DC deficiencies in Chronic Lymphocytic Leukemia (CLL).

Abstract Dendritic cells (DCs) function as principal sensors of PAMPs and DAMPs, connecting the innate and adaptive arms of the immune system. DC subset deficiencies have been observed in untreated CLL patients compared to age-matched healthy controls. Studies investigating the specific DC subsets that differ in function and localization; conventional DCs (cDCs), plasmacytoid DCs (pDCs), and Axl +Siglec-6 +(AS) DCs are lacking in CLL. In mice, DC development and maintenance is highly dependent on the Flt3/Flt3L signaling axis. Activated T cells are believed to be the major source of Flt3L to DCs. Previous studies have reported elevated Flt3L in the serum of untreated CLL patients. Interestingly, increased serum levels of Flt3L have been associated with DC deficiencies, as DCs are believed to be the principle immune cell requiring this cytokine. This study will utilize extensive flow cytometry panels to investigate the prevalence and functional status of DCs in untreated CLL patient blood, including cDC1, cDC2, pDCs, and AS DCs. In addition, we will interrogate the ability of CLL-derived exhausted T cells to provide Flt3L upon activation.

Functional humoral and cellular response of monovalent COVID-19-vaccines against Omicron BA.2 variant of SARS-CoV-2 in patients with chronic lymphocytic leukemia

We designed a prospective study to evaluate the humoral (using a surrogate virus neutralization test) and cellular (using an IFN-γ ELISpot) immune response among patients with chronic lymphocytic leukemia (CLL) against Wuhan-Hu-1 and Omicron BA.2 strains of SARS-CoV-2, after mRNA-based vaccination. The proportion of patients with a functional humoral response was higher among untreated CLL patients compared to treated CLL patients against both Wuhan-Hu-1 and Omicron BA.2 after the second and the third dose of vaccination, and at 12 months after the first dose. The proportion of positive cellular response against the peptide pool covering the full-length Wuhan-Hu-1 spike protein was similar between untreated and treated CLL patients at all three timepoints. The cellular response to the mutated regions of BA.2 spike protein was lower than the response to the corresponding regions in the ancestral spike after the second dose, but this difference was eliminated after the third dose.

EVALUATION OF CD38 ANTIGEN EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE EXPERIENCE OF CLINICAL IMMUNOLOGY DEPARTMENT, NATIONAL RESEARCH CENTER FOR RADIATION MEDICINE, NATIONAL ACADEMY OF MEDICAL SCIENCES OF UKRAINE

Summary. CD38 antigen is a negative prognostic marker of chronic lymphocytic leukemia (CLL). The question of what cut-off level of CD38 antigen expression should be considered as prognostically significant, remains debatable. Aim: to evaluate the optimal cut-off level for CD38 antigen in primary untreated CLL patients for the prognosis of disease and its association with the mutational status of immunoglobulin heavy chain genes (IGHV genes). Objects and methods: flow cytometric and molecular-genetic analysis (mutational status of IGHV genes using polymerase chain reaction followed by direct sequencing) were performed in the groups of 106 primary previously untreated CLL patients. Time-to-treatment, progression-free survival and overall survival were evaluated using the Kaplan-Meier method and Cox regression. Results: The prognostic value of cut-off level CD38 antigen expression ≥ 20% or ≥ 30% does not differ significantly. Lowering of cut-off level to 7% or 5% was unsignificant. The specificity of the evaluation of the mutational status of IGHV genes based on CD38 antigen expression was low, since CD38 expression in almost half of CLL cases with unmutated IGHV genes not differ from CLL cases with mutated IGHV genes. Conclusion: the obtained data confirmed the prognostic value of CD38 antigen expression in patients at the initial stages of disease (A0-AI), primarily for assessing the duration of overall survival.

Bcl-2 Inhibition Induces a Synergistic Effect in Combination with Doxorubicin in Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL), a common neoplastic disease, is associated with the accumulation of B-lymphocytes in the hematopoietic organs. A main characteristic of CLL cells is the failure to undergo apoptosis, thus resulting in resistance to several chemotherapeutics. Doxorubicin (DOX), an anthracycline widely used for treating various neoplasms including CLL, induces apoptosis via several mechanisms. Despite this, CLL cells become resistant to DOX. A major protein known as B-cell lymphoma-2 (Bcl-2), known to exert direct anti-apoptotic effects on the cell, is reported to be overexpressed in CLL cells. Methods: We aimed to silence the Bcl-2 gene by siRNA. Mononuclear cells were isolated from the peripheral blood and bone marrow of eleven untreated CLL patients by Ficoll-Paque. To transfect cells, we used Lipofectamine. Bcl-2 expression was investigated using qRT-PCR. Next, we studied the effect of Bcl-2 silencing in combination with DOX treatment on the viability of cells by an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Results: Bcl-2 expression was significantly suppressed in CLL cells following siRNA transfection via lipofectamine. Based on the results of MTT, the inhibition of Bcl-2 sensitized CLL cells to DOX treatment. Also, the effect of the treatment was time-dependent. Conclusion: These findings imply that combination therapy of CLL using anti-Bcl-2 siRNA and DOX can be considered a practical therapeutic approach that should be further evaluated in future studies.

Anti-PD-L1/PD-L2 therapeutic vaccination in untreated chronic lymphocytic leukemia patients with unmutated IgHV.

Chronic lymphocytic leukemia (CLL) patients with unmutated immunoglobulin heavy chain (IgHV) are at risk of early disease progression compared to patients with mutated IgHV. As a preventive strategy, we treated 19 previously untreated CLL patients with unmutated IgHV in a phase 1/2 trial (clinicaltrials.gov, NCT03939234) exploring the efficacy and toxicity of a therapeutic cancer vaccine containing peptides derived from programmed death ligand 1 (PD-L1) and ligand 2 (PD-L2), hoping to restore immunological control of the disease. According to the International Workshop on Chronic lymphocytic Leukemia (iwCLL) response criteria, no patients obtained a response; however, during follow-up, one patient had complete normalization of the peripheral lymphocyte count and remained in biochemical remission after a follow-up time of 15 months. At the end of treatment, one patient had progressed, and 17 patients had stable disease. During follow-up with a median time of 23.5 months since inclusion, seven patients had progressed, and eight patients had stable disease. The median time to first treatment (TTFT) from diagnosis was 90.3 months with a median follow-up time of 50.1 months. This apparent favorable outcome in TTFT needs to be investigated in a randomized setting, as our population may have been biased. More than 80% of patients obtained vaccine-specific immune responses, confirming the immunogenicity of the vaccine. The vaccine was generally well tolerated with only grade I-II adverse events. Although there were some signs of clinical effects, the vaccine seems to be insufficient as monotherapy in CLL, possibly due to a high tumor burden. The efficacy of the vaccine should preferably be tested in combination with novel targeted therapies or as a consolidating treatment.

Obinutuzumab plus chlorambucil versus ibrutinib in previously untreated chronic lymphocytic leukemia patients without TP53 disruptions: A real-life CLL campus study.

One of the main issues in the treatment of patients with chronic lymphocytic leukemia (CLL) deals with the choice between continuous or fixed-duration therapy. Continuous ibrutinib (IB), the first-in-class BTK inhibitor, and obinutuzumab-chlorambucil (G-CHL) are commonly used therapies for elderly and/or comorbid patients. No head-to-head comparison has been carried out. Within the Italian campus CLL network, we performed a retrospective study on CLL patients without TP53 disruption treated with IB or G-CHL as first-line therapy. Patients in the G-CHL arm had a higher CIRS score and the worst renal function. The overall response rates between the G-CHL and IB arms were similar, but more complete remissions (CRs) were achieved with G-CHL (p = 0.0029). After a median follow-up of 30 months, the progression-free survival (PFS, p = 0.0061) and time to next treatment (TTNT, p = 0.0043), but not overall survival (OS, p = 0.6642), were better with IB than with G-CHL. Similar results were found after propensity score matching and multivariate analysis. While PFS and TTNT were longer with IB than with G-CHL in IGHV unmutated patients (p = 0.0190 and 0.0137), they were superimposable for IGHV mutated patients (p = 0.1900 and 0.1380). In the G-CHL arm, the depth of response (79% vs. 68% vs. 38% for CR, PR and SD/PD; p < 0.0001) and measurable residual disease (MRD) influenced PFS (78% vs. 53% for undetectable MRD vs. detectable MRD, p = 0.0203). Hematological toxicities were common in the G-CHL arm, while IB was associated with higher costs. Although continuous IB provides better disease control in CLL, IGHV mutated patients and those achieving an undetectable MRD show a marked clinical and economic benefit from a fixed-duration obinutuzumab-based treatment.