Development Of Chronic Lymphocytic Leukemia Research Articles

Interleukin-27 potentiates CD8+ T-cell-mediated anti-tumor immunity in chronic lymphocytic leukemia.

Chronic Lymphocytic Leukemia (CLL) cells are highly dependent on interactions with the immunosuppressive tumor microenvironment (TME) for survival and proliferation. In the search for novel treatments, pro-inflammatory cytokines have emerged as candidates to reactivate the immune system. Among those, IL-27 has recently gained attention, but its effects differ among malignancies. Here, we utilized the Eμ-TCL1 and EBI3 knock-out mouse models as well as clinical samples from patients to investigate the role of IL-27 in CLL. Characterization of murine leukemic spleens revealed that the absence of IL-27 leads to enhanced CLL development and a more immunosuppressive TME in transgenic mice. Gene-profiling of T-cell subsets from EBI3 knock-out highlighted transcriptional changes in the CD8+ T-cell population associated with T-cell activation, proliferation, and cytotoxicity. We also observed an increased anti-tumor activity of CD8+ T cells in presence of IL-27 ex vivo with murine and clinical samples. Notably, IL-27 treatment led to the reactivation of autologous T cells from CLL patients. Finally, we detected a decrease in IL-27 serum levels during CLL development in both pre-clinical and patient samples. Altogether, we demonstrated that IL-27 has a strong antitumorigenic role in CLL and postulate this cytokine as a promising treatment or adjuvant for this malignancy.

P281 CARDIAC RICHTER‘S SYNDROME: A RARE CASE OF CHRONIC LYMPHOCYTIC LEUKAEMIA TRANSFORMATION

Abstract A 71–year–old woman with chronic lymphocytic leukaemia (CLL) presented to our Cardiology clinic complaining of recent–onset chest pain and dysphonia. Physical examination and ECG were normal. A blood test revealed high levels of high–sensitivity troponin T (373 ng/L) and of C–reactive protein (7 mg/dL); blood counts and differential were normal. Transthoracic echocardiography showed a hyperechogenic appearance of the interatrial septum, the interventricular septum and the right ventricular free wall, as well as a mild circumferential pericardial effusion. Cardiac magnetic resonance imaging revealed multiple intracardiac masses involving both ventricles and the interatrial septum, with evidence of oedema and fibrosis. A chest computed tomography showed the presence of multiple cervical, pulmonary, and mediastinal masses, one of which was biopsied through video–thoracoscopy. At the histological examination, the mass mainly consisted of large pleomorphic peripheral CD10–, CD20+/–, CD79a+, PAX5+, BCL2+, BCL6–, c–MYC+ B cells, with high proliferative activity (Ki–67/MIB1+ 80%). A diagnosis of CLL transformation into diffuse large B–cell lymphoma (DLBCL), also known as Richter’s syndrome (RS), was established (Figure). Despite indication for specific haematological chemotherapy, the patient rapidly deteriorated and died few days after the diagnosis. RS is a rare evolution of CLL and of small lymphocytic lymphoma, affecting 2–8% of such patients (transformation <1%/ year), yet is burdened with very poor prognosis (median overall survival 8–16 months). Extra–nodal RS is uncommon, and cardiac involvement has been reported in only 3 cases in the literature (in none with the panel of techniques here used). Despite its rarity, CLL patients with rapidly worsening cardiac symptoms should be suspected of RS transformation involving the heart. The main differential diagnosis of cardiac RS is primary cardiac lymphoma. The characterization of cardiac masses relies on multimodality imaging, but histologic analysis is required to reach a definite diagnosis, which is essential to guide subsequent therapeutic choices.

The Broad Spectrum of TP53 Mutations in CLL: Evidence of Multiclonality and Novel Mutation Hotspots

TP53 aberrations are a major predictive factor of resistance to chemoimmunotherapy in chronic lymphocytic leukemia (CLL), and an assessment of them before each line of treatment is required for theranostic stratification. Acquisition of subclonal TP53 abnormalities underlies the evolution of CLL. To better characterize the distribution, combination, and impact of TP53 variants in CLL, 1,056 TP53 variants collected from 683 patients included in a multicenter collaborative study in France were analyzed and compared to UMD_CLL, a dataset built from published articles collectively providing 5,173 TP53 variants detected in 3,808 patients. Our analysis confirmed the presence of several CLL-specific hotspot mutations, including a two-base pair deletion in codon 209 and a missense variant at codon 234, the latter being associated with alkylating treatment. Our analysis also identified a novel CLL-specific variant in the splice acceptor signal of intron 6 leading to the use of a cryptic splice site, similarly utilized by TP53 to generate p53psi, a naturally truncated p53 isoform localized in the mitochondria. Examination of both UMD_CLL and several recently released large-scale genomic analyses of CLL patients confirmed that this splice variant is highly enriched in this disease when compared to other cancer types. Using a TP53-specific single-nucleotide polymorphism, we also confirmed that copy-neutral loss of heterozygosity is frequent in CLL. This event can lead to misinterpretation of TP53 status. Unlike other cancers, CLL displayed a high proportion of patients harboring multiple TP53 variants. Using both in silico analysis and single molecule smart sequencing, we demonstrated the coexistence of distinct subclones harboring mutations on distinct alleles. In summary, our study provides a detailed TP53 mutational architecture in CLL and gives insights into how treatments may shape the genetic landscape of CLL patients.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL

AbstractDysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5′ untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation–related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

Chronic Lymphocytic Leukemia With Two B-Cell Populations of Discordant Light Chain Restrictions in Individual Patients: Parallel Development of Biclonal B-Cell Neoplasms or Clonal Evolution With Isotype Switch?

To evaluate clinicopathologic characteristics of biclonal chronic lymphocytic leukemia (CLL). Retrospectively analyze clinical data and pathologic features. Ten cases were identified in which flow cytometry demonstrated an abnormal B-cell population with a CLL-like immunophenotype but showed no definitive light chain restriction. All had cytogenetic abnormalities detected, including seven with two CLL-related abnormalities. Four of these showed features suggestive of clonal evolution, all having del(13q) as a "stem-line" abnormality and three showing del(11q) as a "side-line" abnormality. Five (50%) cases demonstrated deleterious NOTCH1 mutations, in contrast to 11.8% in a control group of monoclonal CLL (P < .05). Of the 10 patients, 5 received treatment, with good/partial response in three cases and therapeutic resistance in one case. The median treatment-free survival was estimated at 68 months. Despite a polytypic pattern of light chain expression, the neoplastic nature of biclonal CLL is suggested by a characteristic CLL phenotype and can be confirmed by cytogenetic and genomic analyses. The two clones with discordant light chain isotypes may share a "stem-line" cytogenetic abnormality, suggesting possible clonal evolution. Biclonal CLL is associated with NOTCH1 mutations, which may occur in a small subclone and gradually evolve in clonal size. Genomic analysis on light chain-sorted and/or chronologically collected samples may provide insight into clonal evolution in CLL.

Mesenchymal stromal cell senescence in haematological malignancies.

Acute myeloid leukaemia (AML), chronic lymphocytic leukaemia (CLL), and multiple myeloma (MM) are age-related haematological malignancies with defined precursor states termed myelodysplastic syndrome (MDS), monoclonal B-cell lymphocytosis (MBL), and monoclonal gammopathy of undetermined significance (MGUS), respectively. While the progression from asymptomatic precursor states to malignancy is widely considered to be mediated by the accumulation of genetic mutations in neoplastic haematopoietic cell clones, recent studies suggest that intrinsic genetic changes, alone, may be insufficient to drive the progression to overt malignancy. Notably, studies suggest that extrinsic, microenvironmental changes in the bone marrow (BM) may also promote the transition from these precursor states to active disease. There is now enhanced focus on extrinsic, age-related changes in the BM microenvironment that accompany the development of AML, CLL, and MM. One of the most prominent changes associated with ageing is the accumulation of senescent mesenchymal stromal cells within tissues and organs. In comparison with proliferating cells, senescent cells display an altered profile of secreted factors (secretome), termed the senescence-associated-secretory phenotype (SASP), comprising proteases, inflammatory cytokines, and growth factors that may render the local microenvironment favourable for cancer growth. It is well established that BM mesenchymal stromal cells (BM-MSCs) are key regulators of haematopoietic stem cell maintenance and fate determination. Moreover, there is emerging evidence that BM-MSC senescence may contribute to age-related haematopoietic decline and cancer development. This review explores the association between BM-MSC senescence and the development of haematological malignancies, and the functional role of senescent BM-MSCs in the development of these cancers.

PKCβ Facilitates Leukemogenesis in Chronic Lymphocytic Leukaemia by Promoting Constitutive BCR-Mediated Signalling

B cell antigen receptor (BCR) signalling competence is critical for the pathogenesis of chronic lymphocytic leukaemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKCα function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKCβII upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKCα-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKCα-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKCα-KR expressing cells, coincident with upregulation of PKCβII: supported by activation of key signalling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKCβII inhibitor) treatment of PKCα-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKCβII in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKCα-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKCβ expression facilitates leukemogenesis and identify that BCR-mediated signalling is a key driver of CLL development in the PKCα-KR model.

Clonal Evolution of Chronic Lymphocytic Leukemia with Bimodal CD49d Expression Reveals CD49d Plasticity

Clonal Evolution of Chronic Lymphocytic Leukemia with Bimodal CD49d Expression Reveals CD49d Plasticity

Ezrin Is a Potential Druggable Target in Chronic Lymphocytic Leukemia

Despite development in therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or are refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors, but its role in hematological neoplasms is still poorly explored. There is evidence that EZR plays a key function in cell survival and activation of BCR-mediated signaling in B-cell lymphomas. Here, we uncover the expression of EZR in samples from CLL and healthy donors, and the cellular and molecular effects of the NSC305787, a pharmacological EZR inhibitor, in CLL cellular models. For gene expression studies, EZR mRNA expression data from healthy donors (normal lymphocytes, n = 11) and CLL patients (n = 103) samples were derived from the publicly accessible AmaZonia! database 2008 (discovery cohort). Purified CD19+ cells from peripheral blood samples were collected from 56 CLL patients and 10 age-matched healthy donors from the University Hospital of the Medical School of Ribeirão Preto (validation cohort). For functional and molecular assays, peripheral blood mononuclear cells of 24 CLL patients from the Instituto do Câncer do Estado de São Paulo were used. All samples were obtained after informed consent and protocol approved by the Ethical Committee of the Institution. For functional transcriptomics, RNA-seq data published by Landau et al. were obtained from cBioPortal, and all transcripts were pre-ranked according to their differential expression by comparing samples with high versus low EZR expression using limma-voom package in Galaxy and used for gene set enrichment analysis (GSEA). The MEC-1 cell line was cultured following the recommendations of DSMZ. PBMCs from CLL patients were cultured in RPMI Media 1640 and 10% autologous serum. NSC305787 was used as an EZR inhibitor. Cell viability was determined by MTT assay, apoptosis rates were determined by annexin V/PI labeling and flow cytometry, DNA content was performed by PI labeling and flow cytometry, protein expression was determined by Western Blot assay, and gene expression was evaluated by quantitative PCR. Statistical analyses were performed using GraphPad Prism 8; Mann-Whitney test, Student's t-test, or ANOVA and Bonferroni post-test were used for measurable factors, as appropriate. A value of p < 0.05 was considered statistically significant. EZR was highly expressed in lymphocytes derived from CLL patients (discovery cohort: p = 0.003; validation cohort: p = 0.005). GSEA indicated that high EZR expression was positively associated with relevant signaling pathways associated with CLL development and progression, including p53, PI3K/AKT/mTOR, NFkB, and MAPK (all p < 0.05 and FDR q < 0.05). Of note, pharmacological inhibition of EZR had remarkable antineoplastic effects in primary cells derived from CLL patients, corroborating the potential relevance of this target in disease maintenance. In a CLL cell line, EZR inhibition reduced viability, clonogenicity, cell cycle progression, and induced apoptosis (all p < 0.05). Pharmacological EZR inhibition also reduced activation of the ERK, S6RP, and NFkB, indicating that EZR not only associates, but participates in the activation of these signaling pathways in CLL. In conclusion, our results indicate that EZR is high expressed in CLL and associated with molecular signatures relevant to the development and maintenance of the disease. Pharmacological EZR inhibitions attenuate various cellular and molecular behaviors associated with high risk in CLL and may present a new class of drugs to expand the treatment repertoire of this disease. Supported by: FAPESP, CNPQ, and CAPES.

Exploratory Results of PET-CT and Residual Lymph Node Fine Needle Aspiration of Patients Treated with First-Line Venetoclax and Ibrutinib for CLL/Sll; First Interim Analysis of the Phase 2 HOVON 158/Next STEP Trial

Background: Fixed-duration treatment of chronic lymphocytic leukemia (CLL) with venetoclax combined with a BTK-inhibitor(i) results in prolonged progression free survival (PFS). Undetectable levels of minimal residual disease (uMRD) reach an early plateau and are highly predictive for PFS in case of venetoclax in combination with anti-CD20, but kinetics are different when combined with a BTK-i. MRD-levels mirror disease activity in blood (PB) and bone marrow but much less of lymph node (LN) disease, the suspected critical site of CLL proliferation and development of resistance. Although regular CT-scans do provide information on tumor-bulk, there is a high uncertainty concerning the bio-clinical significance of the frequently observed minimally enlarged LN. The NEXT-STEP trial evaluates the efficacy of adding 6 cycles ibrutinib and obinutuzumab for treatment naive patients who do not achieve complete remission (CR) and uMRD after 3 cycles of ibrutinib lead-in and 12 cycles of combined ibrutinib and venetoclax. The first preplanned interim analysis evaluates safety and efficacy for the first 30 eligible patients until the end of cycle 8. In addition, exploratory analyses of 18F-fluorodeoxyglucose (FDG) PET-CT as well as presence of CLL cells in residual LN by fine-needle aspirations (FNA) were performed. We here present this interim analysis. Methods: HOVON 158/NEXT STEP trial is an open-label, single arm phase 2 study. Eligible patients with untreated CLL or small lymphocytic lymphoma (SLL) receive three 28 days cycles of ibrutinib followed by 12 cycles of combined ibrutinib and venetoclax. IwCLL response and MRD bone marrow are assessed after cycle 15. Patients achieving CR with uMRD4 (8-colour flow cytometry, <10-4) stop treatment. Intensification with an additional 6 cycles of ibrutinib combined with obinutuzumab is provided for patients with MRD positivity and/or less than CR. After cycle 8 an exploratory analysis is done with locally assessed Deauville score (DS) on PET-CT. In addition, if at end of cycle 8 a LN > 1.5 cm is present on physical examination or CT scan and located cervical, axillar or inguinal (for safe accessibility), a fine needle aspiration (FNA) by ultrasound is requested. Obtained cells are centrally analyzed by flow cytometry. Results: Median age was 69 years (range 34-85), 70% were male. One patient had SLL. CIRS score > 6 was found in 6 patients (20%). Median eGFR was 65 ml/min (range 39-132). Median lymphocyte count was 157.3 x 109/l (range 2.50-475). CLL-IPI score was low and intermediate in one (3%) and 6 (20%) and high and very high in 18 (60%) and one (3%) patients, respectively. Del (17p)/TP53 mutation was found in 3 patients (10%). Genomic complexity (≥ 3 aberrations) was found in 3 patients (10%). Overall response rate based on PB and CT scan after cycle 8 was 100%. Levels of responses are depicted in table 1. One patient discontinued therapy during the first 8 cycles, due to adverse events. No progression events or death have occurred and no new safety signals were reported at interim analysis. Of all the PET-scans at baseline 13 (43%) demonstrated an increased uptake (DS4) and 5 (17%) uptake > mediastinal blood pool (DS3). In all but one patient DS decreased after 8 cycles; 1 (3%) remainded DS4, 2 (7%) had DS3, 9 (31%) had uptake equal or below mediastinal blood pool (DS2) and 16 (55%) had no uptake (DS1). In 1 patient PET was not performed. Of 13 patients with enlarged LN after cycle 8, FNA could be done in 9 patients, of which 5 contained sufficient number of cells for analysis. FNA was performed in all 3 patients with DS3-4 because of remaining enlarged LN. In all cases, persistent nodular CLL cells were detected by flow cytometry. In 4 of the 5 patients, MRD levels were higher in the LN as compared to PB. The patient with DS4 demonstrated the highest CLL cells percentage in FNA (Figure 1). Conclusion: Three cycles of ibrutinib followed by 5 cycles of ibrutinib and venetoclax results in tumor bulk reduction with a decrease in metabolic uptake on PET-CT in all but one patient. We demonstrated persistent CLL cells by flow cytometry in enlarged LN, suggesting residual LN serve as a sanctuary of CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evolutionary History of Transformation from Chronic Lymphocytic Leukemia to Richter Syndrome

Richter syndrome (RS), an aggressive lymphoma that develops in patients with chronic lymphocytic leukemia (CLL), is a striking example of histologic transformation. While recent advances have transformed the treatment landscape of CLL, RS remains associated with dismal overall survival and understanding of the genetic factors driving evolution of CLL to RS is limited. To decipher the genetics underlying this transformation, we performed an integrative analysis of whole exome (WES) and genome sequencing (WGS) data generated from matched RS and CLL samples from a discovery cohort of 52 patients with RS of diffuse large B cell lymphoma (DLBCL) histology followed by WES analysis of a validation cohort of 45 independent RS cases. Through computational deconvolution of CLL and RS clones, we constructed phylogenetic relationships and traced evolution of CLL to RS, confirming both clonal related (87%) and unrelated RS (13%). In addition to identifying recognized RS-risk genetic lesions, we discovered novel putative somatic driver genes (including IRF2BP2, SRSF1, B2M, DNMT3A, CCND3), copy number alterations (sCNAs) beyond CDKN2A/B loss and MYC gain (including del(15q15)[MGA], del(7q36.2)[EZH2, KMT2C, POT1], gain(1q23.1)[MCL1, NOTCH2] and arm level loss of 1p, 6q, 9p and 9q), whole genome duplication (WGD), and chromothripsis. Additional validation was obtained through re-analysis of 14 RS whole genomes (Klintman et al., Blood, 2021). Overall, our analysis highlighted alterations of NOTCH, DNA damage response and MAPK pathways as frequently preexisting in CLL. Alterations in pathways of MYC signaling, epigenetics, interferon/inflammatory signaling, cell cycle deregulation and immune evasion were newly occurring at transformation. To assess the degree of similarity between RS and DLBCL, non-negative matrix factorization (NMF) clustering was performed on the 97 RS WES samples and 304 previously characterized DLBCL samples (Chapuy et al., Nature Medicine, 2018) based on identified RS and known DLBCL drivers. The majority of RS (75 of 97) clustered separately from DLBCL, while 7 of 8 clonally unrelated RS cases clustered with DLBCL (P=6.75x10-6), with membership across the DLBCL molecular clusters. To further examine RS, NMF clustering was performed across the 97 RS samples, and 5 robust genomic subtypes were identified: RS-1: WGD; RS-2: Trisomy 12 with NOTCH1/SPEN mutations; RS-3: Mut-TP53 and Mut-NOTCH1; RS-4: Mut-EGR2 and Mut-SF3B1; and RS-5: TP53-altered RS without Mut-NOTCH1. Supervised and unsupervised transcriptome analysis of 36 of the 97 RS samples supported the identified genomic subtypes. The less-genome altered RS subtypes (RS-2, RS-4) had improved overall survival (3.3, 11.3, 5.0, 16.7 and 4.0 months for clonally related RS in subtypes 1-5, respectively; P = 0.0082). To investigate the stepwise evolution of CLL to RS at high resolution, single-cell RNA-sequencing was performed on diagnostic biopsy samples from 5 RS patients. We developed a novel tool, CNVSingle, to infer allele specific single-cell sCNAs that enabled identification of the single-cell clusters representing distinct CLL and RS genetic subclones as well as intermediate or transitional evolutionary states. Genetic changes often immediately preceded the transcriptional shifts towards RS and increasing chromothripsis was again observed as a hallmark of RS. RS cells displayed gene expression enriched in pathways of MYC activation and cell cycle, in line with similar analyses on paired RS and CLL bulk transcriptomes. Given the numerous RS-associated genomic features identified by our study, we assessed the feasibility of non-invasive detection of RS events in cell-free DNA (cfDNA) extracted from RS patient plasma by ultra-low pass-WGS sequencing of 46 plasma samples from 24 RS patients. Indeed, RS-specific sCNAs, chromothripsis, RS-specific driver mutations and whole genome doubling were detected in plasma cfDNA, distinct from circulating CLL. This included detection of RS in cfDNA of 6 of 8 patients (75%) at RS diagnosis and 2 of 7 (29%) patients within 10 months preceding RS diagnosis. In conclusion, we report molecular characterization of the largest RS cohort to date (111 patients), demonstrate that RS is genetically distinct from DLBCL and provide novel genetic and prognostic insights into this aggressive malignancy, with potential for clinical translation towards a more precise RS diagnosis.

Ezrin is highly expressed and a druggable target in chronic lymphocytic leukemia

AimsDespite the development of therapeutic strategies for chronic lymphocytic leukemia (CLL), most patients remain incurable, relapse, or refractory to current treatments, indicating the need to expand the antineoplastic repertoire for this disease. Ezrin (EZR) is a known oncogene in solid tumors and plays a key role in cell survival and BCR-mediated signaling activation in B-cell lymphomas. However, its role in hematological neoplasms remains poorly explored. Main methodsThe present study assessed EZR expression in samples from CLL patients and healthy donors and evaluated the cellular and molecular effects of a pharmacological EZR inhibitor, NSC305787, in CLL cellular models. Key findingsEZR was highly expressed and positively associated with relevant signaling pathways related to CLL development and progression, including TP53, PI3K/AKT/mTOR, NF-κB, and MAPK. NSC305787 reduced viability, clonogenicity, and cell cycle progression and induced apoptosis in CLL cells. Pharmacological EZR inhibition also attenuated ERK, S6RP, and NF-κB activation, indicating that EZR not only associates with but also activates these signaling pathways in CLL. Ex vivo assays revealed that the EZR inhibition-induced cell viability reduction was independent of molecular risk and the Binet stage. SignificanceOur study provides insights into EZR as a pharmacological target in CLL, shedding light on a novel strategy for treating this disease.

NOTCH1 Signalling: A key pathway for the development of high-risk chronic lymphocytic leukaemia.

NOTCH1 is a cell surface receptor that releases its intracellular domain as transcription factor upon activation. With the advent of next-generation sequencing, the NOTCH1 gene was found recurrently mutated in chronic lymphocytic leukaemia (CLL). Here, virtually all NOTCH1 mutations affect the protein’s PEST-domain and impair inactivation and degradation of the released transcription factor, thus increasing NOTCH1 signalling strength. Besides sequence alterations directly affecting the NOTCH1 gene, multiple other genomic and non-genomic alterations have by now been identified in CLL cells that could promote an abnormally strong NOTCH1 signalling strength. This renders NOTCH1 one of the key signalling pathways in CLL pathophysiology. The frequency of genomic alterations affecting NOTCH1 signalling is rising over the CLL disease course culminating in the observation that besides TP53 loss, 8q gain and CDKN2A/B loss, NOTCH1 mutation is a hallmark genomic alteration associated with transformation of CLL into an aggressive lymphoma (Richter transformation). Both findings associate de-regulated NOTCH1 signalling with the development of high-risk CLL. This narrative review provides data on the role of NOTCH1 mutation for CLL development and progression, discusses the impact of NOTCH1 mutation on treatment response, gives insight into potential modes of NOTCH1 pathway activation and regulation, summarises alterations that have been discussed to contribute to a de-regulation of NOTCH1 signalling in CLL cells and provides a perspective on how to assess NOTCH1 signalling in CLL samples.

CLL-299 Novel Missense Single Nucleotide Polymorphisms of Human CD38 Gene Allied to Chronic Lymphocytic Leukemia: A Bioinformatics Approach

B-cell chronic lymphocytic leukemia (CLL) is a hematological malignancy that develops from abnormal lymphoid stem cells in the bone marrow and secondary lymphoid tissue (B-lymphocytes). One of the significant contributors to the pathogenesis of CLL is a mutation of CD38 protein. CD38 is a multifunctional ectoenzyme expressed in B-lymphocytes, with a poorly understood in the pathogenesis of CLL. Here we aimed to bioinformatically appraise CD38 missense single nucleotide polymorphisms allied to CLL and to predict their effect on the structure and function of CD38 protein. A literature review identified two SNPs in the CD38 gene that had been discovered by PCR of patients with CLL. Functional analysis by SIFT, Polyphen2, and PROVEAN revealed nine deleterious SNPs in the CD38 coding region. These SNPs were further analyzed by SNAP2, SNP@GO. PMut, STRING, and other software packages which confirmed nine of those SNPs to be truly pathogenic. Stability analysis performed using I-Mutant identified seven SNPs that were predicted to decrease the stability of CD38 and two SNPs predicted to increase it; while MUpro identified eight SNPs that were predicted to decrease the stability of the protein and one SNP that was predicted to increase it. Physiochemical analysis was done using Project HOPE to visualize the most significant SNPs using the Chimera software. Finally, the following SNPs (S193C, D147G, and P98L) were predicted as deleterious in relation to the structure and function of CD38 protein by most of the software used in this study. We reported three novel driver SNPs in the CD38 gene that could result in deleterious effects on protein structure. This genetic variation might eventually alter the molecular functions of CD38 protein implicated in the development of CLL.

Unravelling soluble immune checkpoints in chronic lymphocytic leukemia: Physiological immunomodulators or immune dysfunction.

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape.

Clonal evolution in chronic lymphocytic leukemia is associated with an unmutated IGHV status and frequently leads to a combination of loss of TP53 and TP53 mutation.

Chromosomal abnormalities and gene mutations determine the prognosis of patients with chronic lymphocytic leukemia (CLL). Genetic lesions can be acquired by clonal evolution (CE) likely correlating with clinical progression. Samples of 169 CLL patients were analyzed for cytogenetic clonal evolution (CCE) and CE affecting the genes TP53 and SF3B1. Moreover, the mutational status of IGHV and the clinical outcome was evaluated. CCE was observed in 35% of CLL patients. The most frequently gained cytogenetic aberration was a deletion of TP53. Acquired TP53 deletion was more frequent in patients with SF3B1 mutations compared to those without (19% vs. 7%). CCE showed a tendency to occur more frequently in patients with an aberrant karyotype at first investigation than in patients with a normal karyotype. In 73% of patients with CCE (p = 0.002) and 92% of patients with CE affecting the genes TP53 and SF3B1 (p < 0.001) an unmutated IGHV status was present. CCE and CE affecting the genes TP53 and SF3B1 were significantly associated with each other (p < 0.001). In 7% of patients, CE resulted in the co-occurrence of TP53 deletion and TP53 mutation resulting in a significantly shorter overall survival. The most frequently gained cytogenetic aberration during CCE was a deletion of TP53, which was associated with SF3B1 mutations. Moreover, CCE was associated with an unmutated IGHV status. Our results indicate the importance of re-evaluation of the TP53 status during the course of the disease to ensure correct treatment guidance.

Super-Enhancer-Associated nine-gene prognostic score model for prediction of survival in chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is a type of highly heterogeneous mature B-cell malignancy with various disease courses. Although a multitude of prognostic markers in CLL have been reported, insights into the role of super-enhancer (SE)–related risk indicators in the occurrence and development of CLL are still lacking. A super-enhancer (SE) is a cluster of enhancers involved in cell differentiation and tumorigenesis, and is one of the promising therapeutic targets for cancer therapy in recent years. In our study, the CLL-related super-enhancers in the training database were processed by LASSO-penalized Cox regression analysis to screen a nine-gene prognostic model including TCF7, VEGFA, MNT, GMIP, SLAMF1, TNFRSF25, GRWD1, SLC6AC, and LAG3. The SE-related risk score was further constructed and it was found that the predictive performance with overall survival and time-to-treatment (TTT) was satisfactory. Moreover, a high correlation was found between the risk score and already known prognostic markers of CLL. In the meantime, we noticed that the expressions of TCF7, GMIP, SLAMF1, TNFRSF25, and LAG3 in CLL were different from those of healthy donors (p < 0.01). Moreover, the risk score and LAG3 level of matched pairs before and after treatment samples varied significantly. Finally, an interactive nomogram consisting of the nine-gene risk group and four clinical traits was established. The inhibitors of mTOR and cyclin-dependent kinases (CDKs) were considered effective in patients in the high-risk group according to the pRRophetic algorithm. Collectively, the SE-associated nine-gene prognostic model developed here may be used to predict the prognosis and assist in the risk stratification and treatment of CLL patients in the future.

Abstract A13: Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome

Abstract Richter syndrome (RS), an aggressive lymphoma that develops in patients with chronic lymphocytic leukemia (CLL), is a striking example of histologic transformation. While recent therapeutic advances have transformed the treatment landscape of CLL and lymphoma, RS remains associated with dismal overall survival. Despite an advanced genomic and molecular characterization of CLL over the past decade, the current understanding of the genetic factors driving evolution of CLL to RS is limited. To decipher the genetics underlying this transformation, we have performed an integrative analysis of exome, genome and transcriptome data generated from matched RS and CLL samples from a discovery cohort of 53 patients with newly diagnosed RS of DLBCL histology. Through computational deconvolution of CLL and RS clones, we constructed phylogenetic relationships and traced evolution of CLL to RS, confirming both clonal related (87%) and unrelated cases (13%). In addition to identifying recognized RS-risk genetic lesions, we discovered novel RS-specific alterations, including 5 putative somatic driver genes (IRF2BP2, SRSF1, B2M, DNMT3A and EZH2), frequent copy number alterations beyond del(9p21)(CDKN2A/B), (including amp(7q21.2) (CDK6), amp(9p24) (PDL1/L2), and amp(1q23)(MCL1)), and recurrent whole genome duplication and chromothripsis. Integration of exome and genome sequencing data led to the identification of distinct molecular subtypes of RS with prognostic importance. To confirm these molecular subtypes, a validation cohort of 47 RS cases has been assembled with paired exome and transcriptome data. To further investigate the stepwise clonal evolution of CLL to RS, we performed single-cell RNA-sequencing on biopsy samples obtained at diagnosis from 5 individuals with clonally related transformation. Using a novel tool, CNVSingle, we inferred allele specific single-cell copy number alterations that enabled identification of the single-cell clusters representing distinct CLL and RS genetic subclones as well as intermediate, or transitional, evolutionary states. RS cells displayed gene expression enriched in pathways of MYC targets and cell cycle, in line with similar analysis on bulk transcriptomes. Finally, by ultra-low pass (ULP)-WGS sequencing of plasma samples from RS patients, we demonstrate detection of RS tumor DNA in plasma months prior to initial clinical diagnosis (n=3 of 6) or post-allogeneic stem cell transplant relapse (n=2 of 2). cfDNA is thus a promising tool for early detection of emerging RS and RS relapse as well as for non-invasive detection surrounding diagnosis. Altogether, our study defines RS-specific alterations and provides a molecular definition of RS, identifies distinct genetic subtypes of RS with prognostic significance, traces the evolutionary path to RS and suggests future strategies for improved detection. Citation Format: Erin M Parry, Ignaty Leshchiner, Romain Guieze, Connor Johnson, Eugen Tausch, Sameer A Parikh, Camilla K Lemvigh, Conor Messer, Filippo Utro, Chaya Levovitz, Kahn Rhrissorrakrai, Matthew S Davids, Julien Broseus, Shuqiang Li, Ziao Lin, Binyamin A Knisbacher, Christof Schneider, Laura Z Rassenti, Thomas J Kipps, Nitin Jain, William Wierda, Florence Cymbalista, Neil E Kay, Kenneth J Livak, Brian P Danysh, Chip Stewart, Donna Neuberg, Jennifer R Brown, Laxmi Paridi, Stephan Stilgenbauer, Gaddy Getz, Catherine Wu. Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A13.

CircRIC8B regulates the lipid metabolism of chronic lymphocytic leukemia through miR199b-5p/LPL axis

ObjectiveCircular RNAs (circRNAs) play a critical role in the modulation of tumor metabolism. However, the expression patterns and metabolic function of circRNAs in chronic lymphocytic leukemia (CLL) remain largely unknown. This study aimed to elucidate the role of circRNAs in the lipid metabolism of CLL.MethodsThe expression and metabolic patterns of circRNAs in a cohort of 53 patients with CLL were investigated using whole transcriptome sequencing. Cell viability, liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis, lipid analysis, Nile red staining as well as triglyceride (TG) assay were used to evaluate the biological function of circRIC8B in CLL. The regulatory mechanisms of circRIC8B/miR-199b-5p/lipoprotein lipase (LPL) axis were explored by luciferase assay, RNA immunoprecipitation (RIP), qRT-PCR, and fluorescence in situ hybridization (FISH). CCK-8 and flow cytometry were used to verify the inhibition role of cholesterol absorption inhibitor, ezetimibe, in CLL cells.ResultsIncreased circRIC8B expression was positively correlated with advanced progression and poor prognosis. Knockdown of circRIC8B significantly suppressed the proliferation and lipid accumulation of CLL cells. In contrast, the upregulation of circRIC8B exerted opposite effects. Mechanistically, circRIC8B acted as a sponge of miR-199b-5p and prevented it from decreasing the level of LPL mRNA, and this promotes lipid metabolism alteration and facilitates the progression of CLL. What’s more, ezetimibe suppressed the expression of LPL mRNA and inhibited the growth of CLL cells.ConclusionsIn this study, the expressional and metabolic patterns of circRNAs in CLL was illustrated for the 1st time. Our findings revealed that circRIC8B regulates the lipid metabolism abnormalities in and development of CLL through the miR-199b-5p/LPL axis. CircRIC8B may serve as a promising prognostic marker and therapeutic target, which enhances the sensitivity to ezetimibe in CLL.

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high–B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.