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Ng, Anita, Andrew Shih, Martina Cardillo, Kristin Lynn Sticco, Houman Khalili, Lita Crichton, William Plunkett, Varsha Gandhi, and Nicholas Chiorazzi. "Investigation into Intraclonal Heterogeneity of CXCR4 DimCD5 Bright Chronic Lymphocytic Leukemia Cells Identifies Distinct Activation Signatures." Blood 142, Supplement 1 (November 28, 2023): 3259. http://dx.doi.org/10.1182/blood-2023-187957.
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Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease associated with diverse clinical and molecular profiles, suggesting long-term clonal evolution driven by genetic diversification from a small, often undetected, subset of leukemic cells endowed with superior growth capacities. More importantly, this diversification process enhances the opportunity of CLL subclones to survive selective pressure, such as BTK or PLCG2 mutations that can abolish the effect of BTK inhibitors. While this evolutionary process is inevitable in leukemic cells, therapeutic intervention targeting the cells that have just replicated their DNA, and hence could have accumulated new genomic mutation, would be an effective strategy to significantly delay or abort the emergence of deleterious mutations. In CLL, a series of studies involving consumption of 2H 2O determined that CXCR4 DimCD5 Bright cells (proliferative fraction, PF) of CLL clones contain most of the recently divided cells, identified by the presence of 2H-DNA. This conclusion was supported by finding elevated numbers of Ki67 + and MCM6 + cells in the same fraction, when compared to other CLL cells. Furthermore, PF is superior in: lymphoid tissue homing in xenograft models; activating T cells; the capacity to alter tumor microenvironment (TME) by Th2 skewing; and expression of AICDA which can increase the likelihood of off-target genomic mutations. To permit a focused analysis of PF that would allow us to intercept this evolutionary loop, we characterized the single cell transcriptomes of PF from 4 U-CLL and 4 M-CLL patients immediately prior to ibrutinib treatment. Consistent with previous reports, the majority of PF (herein APC-PF) cells express certain antigen presentation molecules with a gene signature suggestive of T cell regulation and cell-cell interactions. Additionally, we discovered a small subpopulation with an upregulation of MIR155HG, HSP90AB, CCL3, CCL4, MYC and a concomitant MYC activation signature (herein MYC-PF). Since miR-155 expression can be induced by CD40L or B-cell-activating factors, this PF subset may reflect prior BCR ligation and/or T cell co-stimulation. However, the strongest BCR activation signature was found in another PF subpopulation (herein BCR-PF) with an enrichment of germinal center-related genes (e.g., BACH2, PAX5, FOXP1) and CLL-associated molecules ( BCL2, ROR1, MSI2, PLCG2). Furthermore, the BCR-PF cells are least likely to be resting (in G1/G0) compared to others. Collectively, these findings suggest effective BCR engagement, either from the TME or cell-autonomously. Corroborating these findings with our previous studies documenting the CXCR4/CD5 fractions and the expression of protein proliferation markers, our transcriptomic analyses suggest that the BCR-PF may be the most immediate emigrant from the proliferation centers (PC) within CLL lymph nodes (LN). To probe this possibility, spatial transcriptomic analysis was performed on FFPE LN tissues from surgical biopsies from 3 U-CLL and 1 M-CLL patients. Single cell deconvolution was used to determine the relative abundance of cells within the anatomically defined PCs that resemble the three PF subpopulations transcriptomically. Our results suggest that PCs are more enriched in cells resembling BCR-PF, followed by cells bearing the APC-PF signature. To extend on these observations, we have documented that the unique surface markers expressed in these PF subfractions positively correlate with our transcriptomic results. Taken together, these results not only highlighted the molecular heterogeneity of CLL, but also lead to the identification of two PF subpopulations that may provide novel actionable therapeutic targets to significantly halt CLL clonal evolution.
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Julien, Sylvie, Mirjana Radosavljevic, Nathalie Labouret, Sophie Camilleri-Broet, Frederic Davi, Martine Raphael, Thierry Martin, and Jean-Louis Pasquali. "AIDS Primary Central Nervous System Lymphoma: Molecular Analysis of the Expressed VH Genes and Possible Implications for Lymphomagenesis." Journal of Immunology 162, no. 3 (February 1, 1999): 1551–58. http://dx.doi.org/10.4049/jimmunol.162.3.1551.
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Abstract AIDS-associated primary central nervous system lymphomas are late events that have an extremely poor prognosis. Despite different hypotheses, the brain localization of these B cell lymphomas remains an enigma. To better define the cell origin of the lymphomas and the possible role of the B cell receptor (BCR) in the brain localization and/or in the oncogenic transformation, we analyzed the V region genes of the Ig heavy chain expressed by lymphoma cells in five randomly selected patients. After amplifying the rearranged VHDJH DNA by PCR, cloning, and sequencing of the amplified products, we observed that: 1) of the five lymphomas analyzed, four were clearly monoclonal; 2) there was no preferential use of one peculiar VH family or one peculiar segment of gene; 3) the mutation analysis showed that an Ag-driven process occurred in at least two cases, probably before the oncogenic event; and 4) there was no intraclonal variability, suggesting that the hypermutation mechanism is no longer efficient in these lymphoma B cells. Taken together, our results suggest that distinct Ags could be recognized by the BCR of the lymphoma cells in different patients and that, if the Ags are responsible for the brain localization of these B cells bearing mutated BCR, other factors must be involved in B cell transformations in primary central nervous system lymphoma.
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van Bergen, Cornelis A. M., Marvyn T. Koning, Edwin Quinten, Agnieszka Mykowiecka, Julieta Sepulveda, Ramin Monajemi, Ruben A. L. De Groen, et al. "High-Throughput BCR Sequencing and Single-Cell Transcriptomics Reveal Distinct Transcriptional Profiles Associated with Subclonal Evolution of Follicular Lymphoma." Blood 134, Supplement_1 (November 13, 2019): 298. http://dx.doi.org/10.1182/blood-2019-130508.
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Objectives: Follicular lymphoma (FL) typically originates from premalignant mature B cells that carry the founder t(14;18) BCL2 translocation. Mutations in epigenetic modifiers and acquisition of N-glycosylation sites in CDR regions of the B-cell receptor (BCR) are recurrent secondary events in FL pathogenesis. Despite these oncogenic drivers, FL can remain indolent and clinically stable for years. The molecular events driving subclonal evolution into symptomatic progression and eventual transformation to aggressive lymphoma are insufficiently understood. FL cells are frozen in their B-cell development at the germinal center stage and undergo continuous somatic hypermutation mediated by expression of activation-induced deaminase (AID). We aim to identify crucial drivers of subclonal FL evolution by high-throughput mapping at single-cell resolution. Methods: Viable FL cells were isolated and cryopreserved from 23 histologically or immunocytologically confirmed FL samples from 13 patients with informed consent. Full-length VDJ/VJ transcripts were isolated by unbiased template-switching ARTISAN PCR and massive parallel NGS sequencing on the PacBio platform. The clonal primordial FL BCR (pBCR) was reconstructed from unmutated IGV/IGJ sequences with the CDR3 of the least mutated BCR. Since the IgTree program was unable to process the obtained numbers of BCR sequences, we developed the WILLOW algorithm for analysis of BCR evolution based on the principle of maximum parsimony and on distance from the pBCR. Intraclonal BCR variability was quantified by Shannon's diversity index. 5' single cell transcriptomics and VDJ/VJ sequencing was performed on 2 pools of highly purified FL cells from 5 lymph node biopsies on the 10x Genomics platform. Data were deconvoluted based on expressed variants by the Single Cell Sample Matcher (SCSM) algorithm. Clustering based on gene expression profiles was performed by shared nearest neighbour (SNN) modularity optimization within the R Seurat package. Genes whose expression differed significantly (adjusted p<0.05) between clusters were assigned to gene ontology terms. Results: ARTISAN PCR/PacBio NGS yielded a median of 743 full-length VDJ and VJ sequences (range 62-12782) per BCR chain with expected high intraclonal diversity (median 200 subclones, range 15-3301). WILLOW revealed dominant FL subclones with a subclonal hierarchy wherein multiple routes converged to offspring nodes with identical additional mutations rather than tree-like branching (Figure). In serial samples of 4 patients, lymph node biopsies had only marginally higher subclonal diversity than blood or bone marrow samples (p=0,055; Wilcoxon's matched-pairs signed rank test). Overall BCR mutational burden increased over time in sequential biopsies. Two cases of histological FL transformation were dominated by a single subclone (65% and 80% of all VDJ/VJ sequences, respectively) that was rare in the preceding FL BCR network (0.2% and 1.8%). Pooled transcriptomics data from 6050-6500 cells were assigned to individual samples by SCSM and revealed up to seven transcriptional clusters per FL. In 9 of 10 FL, genes assigned to immune function strongly contributed to separation into one or more clusters. Single cell VDJ/VJ sequencing yielded combined heavy and light chain BCR sequences for a median of 502 FL cells per biopsy (range 22 - 1919) that permitted mapping of subclonal evolution by WILLOW based on complete BCR information. Transcriptome clusters were not distributed evenly throughout the WILLOW FL BCR networks but rather statistically associated with distinct major FL subclones. Vice versa, major FL subclones within the same biopsy were distinguished by particular gene expression profiles. Conclusions: WILLOW facilitates mapping of subclonal FL evolution based on high-throughput BCR sequencing. FL evolution proceeds in networks rather than tree-like branching, whereby acquisition of certain combinations of several BCR mutations can occur in parallel in different trajectories. Transcriptomic profiling of single FL cells identifies distinct clusters within a single biopsy. Mapping of these clusters to the FL cell position in the subclonal FL evolutionary network identifies putative mechanisms that are associated with subclonal progression. These mechanisms involve physiological B-cell signalling pathways. Figure Disclosures No relevant conflicts of interest to declare.
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Zaragoza-Infante, Laura, Andreas Agathangelidis, Valentin Junet, Nikos Pechlivanis, Triantafylia Koletsa, Alessio Bruscaggin, Zadie Davis, et al. "Distinct Modes of Ongoing Antigen Interactions Shape Intraclonal Dynamics in Splenic Marginal Zone Lymphoma." Blood 138, Supplement 1 (November 5, 2021): 1330. http://dx.doi.org/10.1182/blood-2021-148722.
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Abstract Almost one-third of all splenic marginal zone lymphoma (SMZL) cases express B cell receptor immunoglobulin (BcR IG) encoded by the IGHV1-2*04 gene. Such cases display a distinctive profile of genomic aberrations (e.g. higher incidence of NOTCH2 and KLF2 mutations) and a more aggressive clinical course compared to SMZL cases utilizing other IGHV genes. Such skewing of the BcR IG gene repertoire implicates antigen selection in SMZL ontogeny. Although the supportive evidence is compelling, it mostly derives from low-throughput approaches, which are inherently limited in their capacity to capture the complexity of the BcR IG gene repertoire. This hinders the comprehensive assessment of the subclonal architecture of SMZL that could offer insight into the dynamics of antigen-IG interactions. Here, we sought to overcome this limitation through a high-throughput immunogenetic investigation of SMZL, focusing on the detailed characterization of somatic hypermutation (SHM) and intraclonal diversification (ID) profiles. Our study included 22 cases utilizing the IGHV1-2*04 gene and 36 cases utilizing other IGHV genes. IGHV-IGHD-IGHJ (IGH) gene rearrangements were PCR-amplified and libraries were sequenced on the Illumina MiSeq platform. Data was analyzed with the IMGT/HighV-QUEST and TRIP software as well as a novel bioinformatics/biostatistics pipeline. Clonotypes were defined as unique combinations of a given IGHV gene+VH CDR3 amino acid (aa) sequence. Only IGH gene rearrangement sequences assigned to the dominant clonotypes of each case were assessed. In detail, all nucleotide variants (nt vars, i.e. all sequences clustered in the same dominant clonotype yet displaying distinct SHM profiles) were identified and further analyzed. Starting from the most abundant nt var, a network was built representing its connections with all other nt vars. For this analysis, we introduce the terms 'most relevant pathway' (MRP) corresponding to the pathway including connected nt vars with the highest total number of IGH sequences; and 'longest mutational pathways' (LMP) corresponding to the pathways with the highest number of nt vars (Fig. 1). Different graph metrics assessed the impact of ID in different SMZL subgroups: the first one focuses on the 'most relevant pathway' and quantifies SHM convergence [ratio of the total number of IGH sequences corresponding to the nt vars of this pathway to the number of IGH sequences in the most abundant nt var]; while the second refers to the length of the 'longest mutational pathways'. Cases lacking additional connected nt vars [length of the LMP=1; 3 IGHV1-2*04 cases and 4 non-IGHV1-2*04 cases] were excluded. Consequently, the analysis included 19 IGHV1-2*04 cases and 32 non-IGHV1-2*04 cases. Significant differences were noted in the SHM and ID profiles between groups; the IGHV1-2*04 group had significantly (p<0.01) higher convergence values ranging from 0.009 to 1.243 (median: 0.102), as opposed to the non-IGHV1-2*04 group (range: 0.002-1.13, median: 0.014), overall suggesting that stronger selective pressures act in SMZL cases expressing the IGHV1-2*04 versus others. Moreover, IGHV1-2*04 cases displayed significantly (p<0.01) longer mutational pathways (length range: 2-6, median: 3) compared to the other group (range: 2-5, median: 2), alluding to more pronounced ID arising due to ongoing SHM. Finally, all mutations leading to aa changes were analyzed in the context of ID networks. More recurrent aa mutations were identified amongst cases with higher levels of convergence. For instance, the VH FR2 M39I change, one of the most prominent recurrent SHMs in the IGHV1-2*04 group, was found in the most abundant nt var in 13/19 IGHV1-2*04 cases, while it was identified in nt vars with extra mutations in another 3 cases. Of interest, it was present at the end of the mutational pathways in these 16 cases, whilst in the other group it was present only in one case using the IGHV1-2*02 gene, and absent in the rest (p<0.01). In conclusion, in the first large-scale high-throughput immunogenetic analysis of SMZL we provide strong evidence for more pronounced antigenic pressure in cases utilizing IGHV1-2*04 versus other IGHV genes. Our findings highlight a unique subclonal architecture for IGHV1-2*04 SMZL and corroborate the hypothesis that this group may represent a distinct molecular variant of SMZL. Figure 1 Figure 1. Disclosures Rossi: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Verastem: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Cellestia: Honoraria, Research Funding. Chatzidimitriou: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Stamatopoulos: Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.
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Yan, Xiao J., Wentian Li, Sophia Yancopoulos, Igor Dozmorov, Carlo Calissano, Sonia Marsilio, Rajendra N. Damle, et al. "Gene Expression Profiles Document That Recently- and Previously-Divided CLL Fractions Represent a Continuum but Suggest Differing Modes of Activation for These Fractions in U-CLL and M-CLL." Blood 120, no. 21 (November 16, 2012): 317. http://dx.doi.org/10.1182/blood.v120.21.317.317.
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Abstract Abstract 317 By using reciprocal densities of surface membrane CXCR4 and CD5, chronic lymphocytic leukemia (CLL) B cells can be divided into 3 fractions indicating time since last division (proliferative, intermediate, and resting). It has been suggested that cells in these fractions represent a continuum from resting to intermediate to proliferative. In this study, we made intraclonal gene expression profile (GEP) comparisons of these fractions from 17 CLL patients to try to confirm this notion and interclonal comparisons between U-CLL and M-CLL patients to determine if pathways involved in the actions of these fractions differed between patient subgroups. PBMCs from 8 U-CLL and 9 M-CLL patients were sorted into 3 fractions (CD19+CD3−CD5hiCXCR4lo, PROLIF), (CD19+CD3−CD5intCXCR4int, INTERM), and (CD19+CD3−CD5loCXCR4hi, REST); RNA was purified from each, and gene expression microarrays using Illumina HumanHT12 beadchips performed. To determine differentially expressed genes in intraclonal comparisons, expression value ratios for fractions from each patient were computed, log-transformed, and Student t-test performed using R (www.r-project.org); for interclonal comparisons, raw GEP data between subpopulations were compared: U-PROLIF and M-PROLIF, and U-REST and M-REST. Sets of significant genes (≥1.5 fold change and P<0.01) were analyzed using Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA). Upon plotting intraclonal average log ratios of PROLIF/INTERM vs INTERM/REST, it was clear that gene expression levels changed in the same direction, i.e. PROLIF>INTERM>REST, or PROLIF<INTERM<REST, consistent with a continuum between the 3 fractions. Within this pattern, 36 genes were significant for both plotted ratios. Of these, 29 were overexpressed, along with CD5; CD68, ITGAX, CCND2, CRIP1 and LGALS1 were the highest. Functional analysis using IPA showed these genes to be related to NFkB signaling and cell trafficking. Seven genes (ADARB1, BACH2, CNTNAP2, HRK, RHPN2, PRPML, and RXPA) were significantly downregulated, along with CXCR4. Next we characterized GEP differences between the PROLIF and REST fractions, identifying 390 genes up-regulated in PROLIF and 244 in REST. The top 5 upregulated PROLIF genes were CD68, LY96, ITGAX, CCND2 and CRIP1, and the top 5 REST genes were BACH2, CXCR4, ADARB1, RHPN2 and HRK. Functionally, the upregulated PROLIF genes were related to BCR signaling, cytokines (IFNa, IL12), NFkB, and Akt, whereas the upregulated REST genes related to BCL2, cell death and cell movement. By GSEA, 813/881 gene sets, defined by expression neighborhoods centered on cancer associated genes, were upregulated in the PROLIF with 436 gene sets significant at a false discovery rate (FDR) <10%; 206 sets were significantly enriched with p value <0.01. For the REST, 68/881 gene sets were upregulated, with none significant even at FDR <25%. Finally, we examined PROLIF and REST fractions from U-CLL vs M-CLL patients. In this interclonal analysis, 93 genes were significantly different between U-PROLIF and M-PROLIF. The top 5 in U-PROLIF were MSI2, TGFBR3, TP53I3, RGCC and IGSF3, and the top 5 in M-PROLIF were MTSS1, BACE2, BRI3BP, AP3B1 and UBE2G2. Similarly, there were 125 genes that were significantly different between U-REST and M-REST. The top 5 in U-REST were DUSP26, CLEC2B, MDK, and EGR2 and in M-REST were NAPSA, RAB24, TARDBP, KCNN4 and ADD3. Interestingly, U-PROLIF and M-PROLIF differed in pathway assignments, with upregulated genes in U-PROLIF contributing to cell signaling and activation, particularly implicating Akt, ERK and P38MAPK. The intraclonal gene GEP analysis on these 3 fractions confirms that CLL clones contain a spectrum of cells that transition in a sequential manner from PROLIF to INTERM to REST fractions. Functional analyses show that genes upregulated in PROLIF correlate with cell signaling and proliferation, while genes upregulated in REST relate to cell death. Thus the PROLIF fraction is enriched in recently divided cells that likely exit from lymphoid tissue and the REST in older, less vital cells that either traffic to lymphoid tissue or die. The interclonal analysis implies that the stimuli and/or the responses of cells in the PROLIF and REST fractions differ between U-CLL and M-CLL. This last novel finding suggests either distinct cells of origin or distinct activation pathways for the IGHV-defined CLL subsets. Disclosures: Barrientos: gilead and pharmacyclics research funding: Research Funding.
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Sutton, Lesley-Ann, Efterpi Kostareli, Anastasia Hadzidimitriou, Nikos Darzentas, Athanasios Tsaftaris, Achilles Anagnostopoulos, Richard Rosenquist, and Kostas Stamatopoulos. "Extensive Intraclonal Diversification in a Subgroup of Chronic Lymphocytic Leukemia Patients with Stereotyped IGHV4-34/IGKV2-30 B cell Receptors: Implications for Ongoing Interactions with Antigen." Blood 114, no. 22 (November 20, 2009): 2337. http://dx.doi.org/10.1182/blood.v114.22.2337.2337.
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Abstract Abstract 2337 Poster Board II-314 Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures: No relevant conflicts of interest to declare.
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Packham, Graham, Serge Krysov, Vania Coelho, Peter Johnson, and Freda K. Stevenson. "A “Universal” Lectin-Mediated Interaction Drives B-Cell Receptor Signaling In Primary Follicular Lymphoma Cells." Blood 122, no. 21 (November 15, 2013): 4291. http://dx.doi.org/10.1182/blood.v122.21.4291.4291.
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Abstract B-cell receptor (BCR) signaling has been identified as a critical driver of B-cell malignancies and as a target for therapeutic attack. Clinical responses to novel inhibitors of BCR-associated kinases have been relatively modest in follicular lymphoma (FL) and a more detailed knowledge of BCR function in these cells is required. Surface Ig (sIg) is unusual in FL since variable regions contain N-linked glycosylation sites which are introduced by somatic mutation. These are rarely found in normal B cells, indicating strong positive selective pressure in malignant cells. Remarkably, added sugars terminate with high mannose suggesting a novel function for FL BCRs in binding to, and perhaps receiving stimulation via, microenvironmental mannose-binding lectins. In previous studies we demonstrated that candidate mannose-binding lectins, including DC-SIGN, promote intracellular calcium mobilization in primary FL cells, but not normal B cells. In this work, we characterized in more detail the response of FL cells to DC-SIGN and its inhibition by BCR-targeted kinase inhibitors. Initial studies using immunoblotting demonstrated that, like anti-Ig antibodies, DC-SIGN caused increased phosphorylation of the downstream kinases AKT and ERK in primary FL samples. DC-SIGN treatment also resulted in increased expression of the MYC oncoprotein in a subset of samples. In contrast to FL samples, DC-SIGN did not increase AKT or ERK phosphorylation in normal B cells although anti-IgM induced strong responses in these cells. Overall, responses to DC-SIGN were similar in IgM+ and IgG+ FL samples. Flow cytometry demonstrated that DC-SIGN also increased phosphorylation of proximal signaling kinases (SYK and BTK), as well as phosphorylation of PLCγ2 in FL samples, and that DC-SIGN-induced signaling occurred within the CD19+BCL2+ malignant cells. Flow cytometry also revealed intraclonal variation in responses to DC-SIGN and, like responses to anti-Ig, DC-SIGN responses were strongest in a sub-population of malignant cells with high CD20 expression. Finally, we demonstrated that tamatinib, the active form of the SYK inhibitor pro-drug fostamatinib, significantly inhibited phosphorylation of ERK and PLCγ2 induced by either anti-Ig or DC-SIGN. Overall our results are consistent with the hypothesis that N-linked glycosylation sites, introduced into BCRs by somatic mutation, are selected for in FL since they confer signaling responsiveness following binding of environmental lectins. Like canonical antigen signaling, lectin-mediated signaling via the BCR appears to be susceptible to therapeutic blockade using kinase inhibitors. However, further analysis of this novel lectin-mediated pathway may reveal novel targets for optimal therapeutic attack. Disclosures: No relevant conflicts of interest to declare.
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Sutton, Lesley-Ann, Efterpi Kostareli, Evangelia Stalika, Athanasios Tsaftaris, Achilles Anagnostopoulos, Nikos Darzentas, Richard Rosenquist, and Kostas Stamatopoulos. "Active Crosstalk with the Microenvironment Leading to Clonal Evolution in Chronic Lymphocytic Leukemia with Stereotyped IGHV4–34/IGKV2–30 Antigen Receptors." Blood 120, no. 21 (November 16, 2012): 2878. http://dx.doi.org/10.1182/blood.v120.21.2878.2878.
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Abstract Abstract 2878 We recently demonstrated that intraclonal diversification (ID) in the immunoglobulin (IG) genes of patients with chronic lymphocytic leukemia (CLL) was limited, with the outstanding exception of subset #4 cases (IGHV4–34/IGKV2–30). Subset #4 cases express IgG-switched antigen receptors carrying long VH CDR3s enriched in positively charged amino acid residues (especially arginine), with acidic residues introduced by somatic hypermutation (SHM) in critical positions of both the heavy and light chain variable domains. This group of patients, characterized clinically by an early age at diagnosis and an indolent disease course, exhibited distinctive patterns of intraclonal diversification (ID) within their IG genes. This may be considered as evidence for an ongoing response to active interaction with antigen (Ag), however, the critical question about the precise timing of Ag involvement and its role in clonal evolution remains unknown. To obtain insight into these issues, we conducted a large-scale subcloning study of the IG genes in a total of 514 and 398 subcloned IG heavy and kappa sequences, obtained from overtime samples from 8 subset #4 cases. All non-ubiquitous sequence changes from the germline among subcloned sequences of the same patient from the same timepoint were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences. Overall, all cases carried intraclonally diversified IG genes. Detailed analysis of the topology and characteristics of mutations revealed: i) restricted ID patterns, in the sense of identical mutations at certain VH/VK positions amongst subclones of different cases, e.g. despite glycine at codon 28 (VH CDR1) being mutated in 413/514 (80%) sequences, the only observed substitution was to an acidic residue, ii) ID hotspots, i.e. mutations in certain codons were repeatedly observed during clonal evolution, and iii) predominance of conservative amino acid changes. Furthermore, the analysis of consecutive samples enabled us to trace the diversification of the CLL antibody over time, and describe the level of ID as increasing, decreasing, or complex when a mutation appears, disappears and then re-emerges at a subsequent time point, thereby also revealing which mutations were negatively or positively selected. Consequently, a stepwise accumulation of mutations could be observed with several CMs at an early timepoint becoming ubiquitous mutations, i.e. present in all subcloned sequences of subsequent timepoints. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially, however at later timepoints the minor cluster had often disappeared and hence been selected against. Despite the high intensity of ID, it was remarkable that certain residues such as in the VH FR1 motif responsible for recognizing the I/i NAL epitope remained essentially unaltered (only 6/514 sequences carried alterations at codon 7 VH FR1). In conclusion, this study defines a clear role for Ag selection in the clonal evolution of CLL subset #4. Whilst the critical eliciting Ag cannot be definitively determined, it is tempting to hypothesize that the distinctive modifications introduced by SHM in the stereotyped BcR likely represent a mechanism for negating auto-reactivity mediated by the arginine-rich VH CDR3s and inducing an anergic state which could potentially be re-activated by subsequent (auto)antigenic stimulation as evidenced by ongoing SHM. It remains to be established whether this stimulation is also accompanied by a change in functional status. Disclosures: No relevant conflicts of interest to declare.
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Linley, Adam J., Beatriz Valle-Argos, Andrew J. Steele, Freda K. Stevenson, Francesco Forconi, and Graham Packham. "Increased Reactive Oxygen Species and the B-Cell Receptor in Chronic Lymphocytic Leukemia Signaling." Blood 124, no. 21 (December 6, 2014): 3291. http://dx.doi.org/10.1182/blood.v124.21.3291.3291.
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Abstract Reactive oxygen species (ROS) play important roles in regulating cell signaling, replication and survival. Chronic lymphocytic leukemia (CLL) cells generally contain high levels of mitochondrial-derived ROS compared to normal B cells. However, there is considerable variation in ROS levels between individual samples. Previous studies have demonstrated that chemotherapy may exert an important influence on ROS levels since prior therapy was linked to increased ROS, potentially via accumulation of mitochondrial DNA damage. However, variability in ROS levels is also apparent between samples from untreated patients demonstrating that additional factors influence ROS levels in CLL cells. One additional variable may be signalling via the B-cell receptor (BCR), now recognised as a major determinant of disease behavior and a target for therapeutic attack. BCR signalling appears to be on-going in CLL with the balance between antigen-induced anergy and positive signalling influencing outcome. We therefore investigated whether ROS levels correlated with subsets of diseases defined by IGHV mutation status or extent of anergy, and with outcome. We also investigated the role of ROS in modulating signaling via surface IgM (sIgM) in vitro. Flow cytometric analysis of 33 peripheral blood mononuclear cell samples demonstrated that ROS levels were highly variable between individual CLL patients. As demonstrated previously, mitochondria appeared to be the major source of ROS in CLL cells. ROS levels were higher in M-CLL compared to U-CLL (P=0.01). ROS levels were also higher in samples that had strong features of anergy, including down-modulation of surface IgM (sIgM) expression (P=0.003) and signaling capacity (P=0.001). Importantly, higher levels of ROS were associated with longer time to first treatment (P=0.003). Overall, high levels of mitochondrial-derived ROS appear to be a feature of anergy and are associated with M-CLL and indolent disease. Some patients demonstrated intraclonal variation in ROS production. This was observed in both M-CLL and U-CLL, but was somewhat more prominent in U-CLL. To probe this variation, we investigated expression of CXCR4 in these sub-populations since down-modulation of this receptor “marks” cells which have most recently entered the circulation following tissue-based stimulation. CXCR4low CLL cells contained relatively high levels of ROS compared to “recovered” CXCR4high cells indicating that increased ROS was a consequence of tissue based stimulation. Engagement of the BCR by antigen could be one factor that modulates ROS levels in vivo. We were unable to detect any changes in ROS levels following sIgM stimulation in vitro and therefore investigated effects of the anti-oxidant N-acetylcysteine (NAC) on anti-IgM-induced signaling. NAC significantly inhibited anti-IgM-induced ERK1/2 and AKT phosphorylation demonstrating that ROS are required for optimal signalling via sIgM. NAC also inhibited CXCR4-mediated migration suggesting that ROS are important regulators of multiple receptors in CLL cells. Overall, our results suggest that BCR signaling may be an important modulator of ROS in CLL cells. However, interactions are complex with evidence for both compartmentalization and temporal separation of responses which can only be partly probed with currently available tools. Anergy appears to be associated with increased mitochondrial ROS production, whereas growth promoting BCR signaling may be associated with a transient, localized accumulation at the membrane. CLL cells have increased sensitivity to induction of apoptosis by agents which further enhance ROS and this has been proposed as the basis for novel therapeutic approaches. However, it will be important to consider the multifactorial nature of ROS revealed in our experiments and examine potentially deleterious effects of increased ROS on enhanced BCR signaling. Disclosures No relevant conflicts of interest to declare.
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Baaklini, Sabrina, Alexandre Sarrabay, Camille Barthelemy, Laila Dahbi, Laurine Gil, Noushin Mossadegh-Keller, Jean-Marc Navarro, et al. "A Single Cell Atlas of Diffuse Large B Cell Lymphomas Reveals Distinct Cellular States Predictive of Outcomes." Blood 142, Supplement 1 (November 28, 2023): 848. http://dx.doi.org/10.1182/blood-2023-173929.
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Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-cell lymphoma with high clinical and biological heterogeneity. Despite current effective immunochemotherapy, up to 40% of patients do not respond or develop refractory disease. DLBCL is characterized by two major cell-of-origin (COO) subtypes, germinal center B cell-like (GCB) and activated B cell-like (ABC), with recent research uncovering additional molecular subgroups based on genomic alterations or tumor microenvironment (TME) features. However, current classifiers use bulk or deconvolution profiling which blurs intra-tumor heterogeneity, hindering the discovery of true functional cell states underlying clinical diversity. Here, we constructed a multimodal single-cell atlas of 62 DLBCL cases, integrating single-cell transcriptomes, immune repertoire and genetic features, to decrypt recurrent B cell states and TME ecosystems while identifying clinically relevant prognostic biomarkers (Fig.1). We performed simultaneous 5'-end single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing on 63 DLBCL lymph node biopsies, recovering 109,294 cells after stringent quality controls. These biopsies were obtained from the real-world, clinically-annotated, multicentric CeVi collection and included 43 patient samples at diagnosis (ABC = 17, GCB = 11, Unclassified = 6, Not Evaluated = 9; 21 of which did not achieve event-free survival at 24 months (EFS24) after R-chemotherapy regimens), 11 relapsed/refractory DLBCL and 8 DLBCL transformed from indolent lymphoma. We distinguished malignant B-cells from TME using canonical markers and BCR sequence, exploring heterogeneity in both components. We developed an original bioinformatic approach to define malignant B cell transcriptional archetypes shared across patients. To assess whether subclonal genetics are associated with transcriptional states, we inferred copy number variations (CNV) and studied BCR intraclonal variations of malignant cells. We also integrated TME data (39,674 cells) to quantify subpopulations and clonally expanded T-cell subsets, and performed robust statistical analyses to identify clinically relevant features linked to patient outcomes. To circumvent interpatient heterogeneity in malignant B cells, we defined transcriptional cellular archetypes conserved across patients by combining 3 unsupervised methods: rank-biased overlap meta-clustering, variational autoencoder and canonical correlation analysis integration. We identified 5 cellular archetypes (Arch.1-5): Arch.1 was characterized by plasma cell identity, Arch.2 by MYC and NFKB pathways, Arch.3 by MYC pathway only, Arch.4 by quiescence markers, and Arch.5 by GC markers. Several archetypes co-existed in each tumor sample, and archetype identity was superimposed to the transcriptional profile conferred by COO molecular class as an additional layer of transcriptional heterogeneity. Cell state-specific CNV and BCR subclones were very rare, suggesting that subclonal genetic changes were not the main drivers of malignant B cell plasticity and archetypes. Kaplan-Meier analysis showed a significant negative association between proportion of Arch.4 cells and continuous EFS in our diagnosis cohort. Unsupervised analysis of archetype composition further distinguished ABC patients who achieved EFS24 (enriched in Arch.3 cells) from those with a particularly poor prognosis (enriched in Arch.4 cells). Integrated analysis of the TME revealed three major ecosystems, including one enriched for effector and cytotoxic T cells. In diagnosis samples, pseudo-temporal trajectory analysis revealed the enrichment of clonally-expanded hyperactivated CD8+ T cells expressing canonical exhaustion markers in patients achieving EFS24, suggesting a positive link between the extent of CD8+ T cell differentiation and response to R-CHOP based regimens. Shedding new light on DLBCL biological and clinical heterogeneity, our comprehensive single-cell atlas of DLBCL uncovered recurrent tumor cell states and TME features crucial for stratifying non-responsive ABC patients and guiding treatment decisions. Ongoing single-cell spatial transcriptomics analyses will be used to further validate our findings and elucidate archetype-TME interactions that may drive malignant B cell plasticity, with the potential to uncover novel therapeutic targets.
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Gemenetzi, Katerina, Andreas Agathangelidis, Lesley-Ann Sutton, Elisavet Vlachonikola, Chrysi Galigalidou, Fotis Psomopoulos, Maria Gounari, et al. "Remarkable Functional Constraints on the Antigen Receptors of CLL Stereotyped Subset #2: High-Throughput Immunogenetic Evidence." Blood 132, Supplement 1 (November 29, 2018): 1839. http://dx.doi.org/10.1182/blood-2018-99-119125.
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Abstract Subset #2 is the largest subset carrying stereotyped B cell receptor immunoglobulin (BcR IG) in chronic lymphocytic leukemia (CLL). This particular BcR IG is composed of heavy (HC) and light (LC) chains encoded by the IGHV3-21 and the lambda IGLV3-21 gene, respectively. The clonotypic IGHV3-21 genes display a variable load of somatic hypermutation (SHM), being mostly classified as mutated (M-CLL) but also including unmutated (U-CLL) cases. Subset #2 cases, independently of the SHM status, have a particularly dismal clinical outcome similar to that of patients with TP53 aberrations, although lacking such aberrations. Subset #2 BcR IG display a series of distinctive features, including conservation at certain VH and VL CDR3 positions and recurrent SHMs; as well as a capacity for self-association leading to cell autonomous signaling that is critically dependent on a substitution of Arginine (R) for Glycine (G) introduced by SHM at the lambda VL-CL linker region. These features implicate antigen selection in CLL subset #2 ontogeny. However, the available molecular evidence derives from low throughput immunogenetic analysis, precluding comprehensive assessment of antigenic impact on (sub)clonal composition. Here, we sought to overcome this limitation by performing next-generation sequencing (NGS) of HC and LC gene rearrangements of 20 subset #2 patients. RT-PCR products amplified by the IGHV3-21/IGHJ6 and IGLV3-21/IGLC primer pairs, respectively, were subjected to NGS on the MiSeq Illumina Platform. NGS data was analyzed by a validated bioinformatics pipeline. Rearrangements with identical CDR3 amino acid (aa) sequences were defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. Starting with HCs, we obtained 3,340,508 (mean: 291,751, range: 101,231-186,055) productive reads. On average, each analyzed sample carried 92 distinct clonotypes (range: 71-152), with the dominant clonotype having a mean frequency of 96% (range: 67-99%): in all cases the dominant clonotype was identical to that determined by Sanger sequencing. The dominant clonotype displayed considerable intraclonal heterogeneity with a mean of 5,082 subclones/sample (range: 2,946-11,041). Turning to LCs, we obtained 5,094,045 (mean: 231,547, range: 38,036-507,949) productive reads. LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean 222, range: 156-306). The dominant clonotype had a mean frequency of 96% (range: 74-98%); similar to HCs, it was identical to that determined by Sanger sequencing. Intraclonal heterogeneity was observed in the LCs as well, with a mean of 7,382 subclones/sample (range: 1,946-11,866), hence more pronounced vs their partner HCs. Viewing the entire subset #2 VH or VL CDR3 dataset (i.e. the CDR3 aa sequences from all clonotypes of all cases) as a single entity branching through diversification enabled the identification of 2 distinct VH CDR3 sequences present at varying frequencies in 16 and 13 cases, respectively; and, 3 distinct VL CDR3 sequences present at varying frequencies in all 20 cases: these results allude to important constraints on the composition of the antigen binding site. Focusing on SHM, the following notable observations could be made. (i) The G-to-R substitution at the VL-CL linker was a clonal event in all cases with R being degenerately encoded by different nucleotide sequences; altogether, these findings underscore the seminal role of this recurrent SHM, likely due to mediating self-association. (ii) A recurrent 3-nucleotide deletion was detected in the VH CDR2 of all cases, strongly supporting functional pressure. This change, previously identified by Sanger sequencing as a recurrent SHM in subset #2 (albeit at a frequency of only 25%), was clonal in 4 cases and subclonal in the remainder, where it was present in an average of 105 subclones/sample (range: 1-369). (iii) Certain positions in both the VH and VL domain bore the same aa substitution, mostly at subclonal level: the prime example concerned the G for Serine (S) substitution within the VL CDR3, detected in all samples at a mean frequency of 44.2% (range: 6.3-87%). In conclusion, we provide compelling immunogenetic evidence for functional pressure in the ontogeny of CLL subset #2. On this evidence, subset #2 emerges as perhaps the most striking example of antigen-driven leukemogenesis reported thus far. Disclosures Gemenetzi: Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Hadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.
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Ptacek, Jason, Erik Evensen, Greg Friedland, James Cordeiro, Jodi R. Ware, Charles C. Chu, Rajendra N. Damle, et al. "Single Cell Network Profiling (SCNP) Identifies Altered Signaling Between Patient Risk Groups in B-Cell Chronic Lymphocytic Leukemia (B-CLL)." Blood 120, no. 21 (November 16, 2012): 2876. http://dx.doi.org/10.1182/blood.v120.21.2876.2876.
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Abstract Abstract 2876 Background: B-CLL follows a variable clinical course, with a subset of patients progressing quickly. The reasons for this outcome disparity are not fully understood; however, evidence suggests that B-cell receptor (BCR) signaling is a driving event in disease onset and progression. B-CLL cells also receive survival signals through additional receptors. SCNP is a multiparametric flow cytometry-based assay that measures, quantitatively at the single cell level, changes in intracellular signaling proteins in response to extracellular modulators. This provides a functional measure of pathway activity and intraclonal signaling differences within the larger B-CLL cell population. In prior studies, we observed higher αIgM-induced (→) p-ERK signaling in B-CLL samples from patients who had a shorter time to first treatment (TTFT) (Cesano et al. ASH 2011 Abstract 2834). Herein we examined the feasibility for SCNP to further define patient risk stratification. Objectives: The current study was designed to 1) map signaling profiles in early-stage B-CLL and to 2) identify signaling associations with clinical subgroups defining B-CLL prognosis (IGHV mutational status, cytogenetic risk, CD38 / ZAP-70 expression). Methods: Between 2006 and 2007, peripheral blood mononuclear cells (PBMCs) were collected and cryopreserved from a cohort of 39 untreated B-CLL patients, Rai stage 0 - II, at different time points during their clinical course. Of the 39 samples evaluated; 15 and 20 expressed CD38 (≥30% of cells) and ZAP-70 (≥20% of cells), respectively; 19 were IGHV unmutated (98% cutoff); and cytogenetic risk groups were evenly represented. SCNP analysis quantitatively measured 22 intracellular signaling proteins within CD19+CD5+ B-CLL cells, using a panel of 14 disease-relevant modulators. The Wilcoxon rank-sum test was used to identify signaling associations with CD38 and ZAP-70 expression, cytogenetic risk categories, del17p (includes p53 gene deletion) and IGHV mutational status. Results: Significant associations between patient risk categories and signaling are summarized in Table 1. IGHV unmutated and ZAP-70+ samples showed (1) elevated α-IgM or α-IgD-induced BCR signaling, either alone or in combination, (2) decreased CpGβ → p-ERK induction and (3) increased thapsigargin-induced (Ca2+ signaling) signaling. Of note, CD38+ samples did not show the same associations, but shared with unmutated IGHV samples a higher responsiveness to IFNα and weaker induction of p21 in response to bendamustine. The unfavorable cytogenetic group samples showed increased αIgM→p-ERK and had higher basal p-S6 that further increased with IgD crosslinking. Lack of p21 induction was also associated with unfavorable cytogenetics, which includes deletion of p53 (del17p), a regulator of p21 expression. Conclusions: This is the second, independent SCNP analysis of B-CLL signaling showing decreased bendamustine→p21 and increased αIgM→p-ERK signaling in samples with unfavorable cytogenetics. Further associations with IGHV unmutated status included increased BCR signaling in multiple nodes, altered TLR9 responsiveness, and decreased drug-induced p21. These data support the potential utility of SCNP in: (1) identifying in one assay those patients with a more aggressive form of B-CLL, including both unmutated IGHV and p53 pathway alteration, and (2) identification of patients with signaling profiles that may be more likely to respond to targeted therapies. Disclosures: Ptacek: Nodality, Inc.: Employment, Equity Ownership. Evensen:Nodality, Inc.: Employment, Equity Ownership. Friedland:Nodality, Inc.: Employment, Equity Ownership. Cordeiro:Nodality, Inc.: Employment, Equity Ownership. Ware:Nodality, Inc.: Employment, Equity Ownership. Cesano:Nodality, Inc: Employment, Equity Ownership. Hawtin:Nodality, Inc.: Employment, Equity Ownership.
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Vardi, Anna, Andreas Agathangelidis, Evangelia Stalika, Millaray Marincevic, Maria Karypidou, Achilles Anagnostopoulos, Chrysoula Belessi, Nikos Darzentas, Richard Rosenquist, and Kostas Stamatopoulos. "T Cell Receptor Gene Repertoire Restriction in Chronic Lymphocytic Leukemia with Stereotyped IGHV4–34/IGKV2–30 Antigen Receptors." Blood 120, no. 21 (November 16, 2012): 3908. http://dx.doi.org/10.1182/blood.v120.21.3908.3908.
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Abstract Abstract 3908 Chronic lymphocytic leukemia (CLL) exhibits a remarkably skewed immunoglobulin (IG) gene repertoire mainly evident in the existence of subsets of patients with quasi-identical IGs in their B cell receptors (BcRs), collectively accounting for one-third of CLL patients. BcR stereotypy is strongly suggestive of clonal selection by a restricted set of antigens. However, it is not yet clear at which phase of clonal evolution these antigens act, or whether the stimulation is persistent. Furthermore, the possible role of antigens in the selection and activation of cognate T lymphocytes remains obscure yet highly relevant, given recent data about T cell interactions with CLL B cells and their tolerized behavior. Here, we analyzed the repertoire of T cell receptor β chain genes (TRB) in CLL expressing stereotyped IGHV4–34/IGKV2–30 BcR IGs (subset #4), which exhibit a series of immunogenetic features, such as pronounced intraclonal diversification of IG genes, suggestive of ongoing interactions with (auto)antigens. Furthermore, subset #4 CLL cells have distinctive functional responses to BcR and/or Toll-like receptor triggering, rendering this subset a paradigmatic example for seeking evidence of antigen selection also within the T cell population. We analyzed 18 peripheral blood samples of 12 untreated subset #4 patients (samples from different time points were analyzed in 4 cases). No case had evidence of infection at sampling. PCR amplicons for TRBV-TRBD-TRBJ gene rearrangements (BIOMED2 protocol) were subcloned by transformation into E. coli/TOP10F bacteria and randomly chosen individual colonies were subjected to Sanger sequencing. Only productive rearrangements (n=320, ranging from 14–52/case) were analyzed. All cases were found to carry clusters of identical rearrangements (≥2) corresponding to distinct clonotypes; the number of expanded clonotypes/case ranged from 1–13 (median 5). The relative frequency of each clonotype/case was determined by dividing the number of the corresponding identical sequences by the total number of subcloned sequences analyzed. The frequency of the most expanded (immunodominant) clonotype/case ranged from 8.1–70.4%. Collectively, the frequency of all expanded clonotypes/case ranged from 29.7–93.3%. In 2/4 cases that were analyzed at different time points, at least one clonotype was found to persist. Importantly, cluster analysis of the TRB CDR3 sequences of all cases identified ‘public’ clonotypes: 2 identical clonotypes (TRBV15*02/TRBD1*01/TRBJ2–2*01 and TRBV30*01/TRBD1*01/TRBJ2–2*01) each shared by a pairs of different patients and a highly similar clonotype shared by an additional pair of patients. In conclusion, the present study provides clear evidence of repertoire skewing among T cells in CLL patients belonging to subset #4, strongly supporting antigen selection. The finding of ‘public’ clonotypes raises the possibility that shared antigenic epitopes may be relevant for clonal selection of T cells in different subset #4 cases. Whether the antigens that drive T cell repertoire restriction are identical/related to those implicated in the selection of CLL progenitors of subset #4 or even the malignant cells themselves or whether they are tumor-associated antigens remains to be clarified. Disclosures: No relevant conflicts of interest to declare.
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Mazzarello, Andrea Nicola, Mark Fitch, Anita Ng, Sabreen Bhuiya, Esha Sharma, Andrew Shih, Martina Cardillo, et al. "Analyses of the Kinetics and Phenotype of Multiple Intraclonal CXCR4/CD5 B Cell Subsets Suggest Differences in Life Cycle Transitioning in CLL." Blood 138, Supplement 1 (November 5, 2021): 2622. http://dx.doi.org/10.1182/blood-2021-154224.
Full textAbstract:
Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease so that defining the dynamic features of the clone and its intraclonal subpopulations are essential to understand disease pathogenesis and to develop novel, effective therapies. For instance, because cell division is linked with new mutations, the ability to preferentially select cells that recently divided allows studying the subpopulation(s) most likely responsible for disease progression and resistance to therapies. The intraclonal kinetics of CLL B cells have been studied in clonal subgroups defined by reciprocal surface levels of CXCR4 and CD5. In that model, three fractions are identified: recently divided "proliferative" (PF; CXCR4 DimCD5 Bright); "intermediate" (IF; CXCR4 IntCD5 Int) and "resting" (RF; CXCR4 BrightCD5 Dim). Here, we have expanded the examination of subpopulations differing for time since last division ("age"). Unmanipulated CLL cells studied ex vivo from 10 patients who drank 2H 2O for 4 weeks were sorted by the relative densities of CXCR4 and CD5 to isolate the formerly identified PF, IF and RF as well as two fractions not previously characterized, "Double Dim" (DDF: CXCR4 DimCD5 Dim) and "Double Bright" (DBF; CXCR4 BrightCD5 Bright). For each fraction, the amount of deuterium incorporated into cellular DNA in vivo was measured. Consistently, the PF contained significantly higher levels of 2H-labeled DNA and higher calculated cell division rates when compared with the RF and IF. Interestingly, the DDF also contained significantly more 2H-labeled DNA compared to the RF; in contrast, the DBF resembled more closely the RF fraction. The overall 2H-incorporation gradient was: PF>DDF>IF>DBF>RF. In CLL, BCR signaling is fundamental, with the amount of membrane (m) IgM associating with signaling competence and disease aggressiveness. Additionally, when engaged independently, mIgM and mIgD can lead to different signaling sequelae. Therefore, we analyzed the 5 subpopulations for the densities of mIgM and mIgD. This showed a distribution similar to that of 2H-DNA incorporation: for IgM: PF=DDF>IF=DBF=RF, and for IgD: PF>DDF>IF=DBF>RF. Accordingly, we next measured 2H-DNA in subpopulations with low, intermediate and high levels of IgM and IgD. This revealed a direct correlation between IG densities and in vivo DNA synthesis, consistent with intraclonal subpopulations with high IGs having divided more recently than those with low IGs. However, these findings are not in line with cell division being primarily initiated by BCR engagement since that would lower mIgM levels. Therefore, we tested if engagement of TLR9 would affect mIG densities on CLL cells. After stimulation of 32 CLL clones with CpG+IL15, anti-IgM+IL4, anti-IgD+IL4, or anti-IgM-IgD+IL4, there was a significant increase in mIGs only after CpG+IL15 activation; each anti-IG stimulation led to downregulation of mIGs. Finally, we questioned the subclonal responsiveness to BTK inhibition in vivo. CLL samples taken from the same patients, before and during ibrutinib treatment, displayed intraclonal changes in mIG densities and cell size, the latter a marker of cellular and metabolic activation also linked with CLL in vivo birth rates. Ibrutinib treatment normalized mIgM and mIgD intraclonal densities and lead to an overall cell size decrease with larger, 2H-enriched and higher mIG density cells being more affected (PF>DDF>IF>DBF>RF). Collectively, these findings suggest that the most recently born cells enter the circulation as the PF from which they transition to either lower CD5 (DDF) or higher CXCR4 (IF and DBF) phenotypes. Each eventually converge as the RF. Moreover, since mIG densities on the more recently divided populations (PF and DDF) are high, the data imply that successful cell division is not solely a consequence of BCR engagement; the involvement of the TLR pathways, concomitantly or in series with BCR signaling, is more consistent with the higher mIG levels. Finally, ibrutinib treatment appears to preferentially target more recently divided cells with high mIG levels. Disclosures Allen: Alexion: Research Funding; Bristol Myers Squibb: Other: Equity Ownership; C4 Therapeutics: Other: Equity Ownership; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees.
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Odabashian, Mariette, Emanuela Carlotti, Shamzah Araf, Jessica Okosun, Francesco Forconi, Freda K. Stevenson, John G. Gribben, Maria Calaminici, and Sergey Krysov. "Immunoglobulin Variable Region Gene Sequences Reveal N-Glycosylation Motifs As an Early and Stable Event in Follicular Lymphoma Pathology." Blood 132, Supplement 1 (November 29, 2018): 4101. http://dx.doi.org/10.1182/blood-2018-99-112397.
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Abstract Introduction: Follicular lymphoma (FL) cells retain expression of a functional B cell receptor (BCR) despite the loss of one Ig allele due to the hallmark t14:18 translocation and ongoing somatic hypermutation (SHM) of the variable genes (V genes) which increases the likelihood of crippling mutations. SHM introduces N-glycosylation (N-gly) motifs within the V genes, a feature exclusively restricted to germinal centre (GC)-derived lymphomas. Oligosaccharides of the high mannose type are added to motifs and interact with calcium-dependent lectins associated with cells of the microenvironment. This activates the BCR signalling pathway and likely contributes to the survival, retention and proliferation of tumor cells in the GC. Determining at what stage of disease evolution N-gly motifs are acquired and their behaviour during progression can ascertain their importance in pathogenesis and their potential as an effective therapeutic target. To achieve this, we analysed motifs within the Ig heavy chain variable gene (IGHV) of tumor-related subclones across temporal FL samples. Method: Genomic DNA from three FL patients taken at different time points of disease progression were analysed. In total, 8 samples were selected, all carrying an IGHV3 rearranged tumor clone. IGHV DNA amplicons were sent for 2x250bp paired-end sequencing using the Miseq Illumina platform (Genewiz, NJ). Tumor-related reads with counts greater than ten were selected following analysis on IMGT/HIGH-V-QUEST. Reads were aligned and unique sequences were assigned as subclones. Additional tumor related reads sequenced on the Roche 454 Life Sciences Genome Sequencer FLX were available (Patients 4 & 5). Subclones were analysed for N-gly motifs and evolutionary pathways were generated using the IgTree program, based on intraclonal SHM profiles and homology of tumor clones to the germline IGHV sequence. Results: The earliest time point samples for Patient's 1, 3, 4 & 5 contained one N-gly site within the IGHV of the MC defined by the largest count number. These sites were conserved in >97% of unique subclones (p<0.0001) despite variations in the nucleotide sequence within the region as a result of ongoing SHM. This conservation included the most mutated subclones which had a mean SHM rate of 16.56%. Conservation was maintained across disease events. Patient 2 contained four N-gly sites located within the CDR1, FR2, CDR2, and FR3 regions. The first three sites were conserved in >97% of subclones in and across disease events, whereas the FR3 site was conserved in 95.5% of the diagnostic subclones and in ~80% of the relapsed and transformed populations. No subclones with loss of all four sites were detected for Patient 2. Patient 5 samples were taken from different anatomical sites with tumor populations acquiring distinct N-gly motifs, suggesting an early divergence in tumor evolution. Despite this, a minor population of motif positive clones are shared, suggesting a trafficking ability of subclones. Subclones with motifs made up ≥99% of the total tumor count, highlighting the motif as a feature of the tumor bulk, with motif negative clones representing a minor population. These negative clones are presumably lost during disease progression as they not shared between events. Evolutionary analysis revealed no additional sites are gained as motif positive clones expand while rare negative subclones cannot reacquire sites and do not undergo further diversification. Conclusion: We report for the first time that acquired N-gly motif sites are a clonal feature in FL disease as seen through their conservation both in the heterogeneous subclonal population and the overall tumor mass. The sites are also retained in progression-associated subclones while rare motif-negative subclones disappear. This suggests that although acquisition of additional driver mutations may dampen the tumor's microenvironment dependency, the motifs and added mannoses may retain functional significance at later stages of disease. The data indicates motifs as being a universal event of the reservoir cell pool responsible for propagating disease episodes. Targeting N-gly sites and their interacting partners may lead to the disruption of an early and vital FL-microenvironment interaction, presumably mediated through the mannose-lectin interaction, reducing relapse rates and progression of disease. Disclosures Forconi: Abbvie: Consultancy; Janssen-Cilag: Consultancy. Gribben:Abbvie: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Cancer Research UK: Research Funding; Wellcome Trust: Research Funding; Acerta Pharma: Honoraria, Research Funding; TG Therapeutics: Honoraria; NIH: Research Funding; Kite: Honoraria; Janssen: Honoraria, Research Funding; Unum: Equity Ownership; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding.
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Roulland, Sandrine, Julie Agopian, Mélanie Briand, Jean-Marc Navarro, Yannick Lecluse, Pascal Gauduchon, Pierre Lebailly, and Bertrand Nadel. "Long-Term Clonal Evolution of Circulating Follicular Lymphoma-Like B Cells in Healthy Individuals: An Early Pathway to Lymphomagenesis." Blood 110, no. 11 (November 16, 2007): 185. http://dx.doi.org/10.1182/blood.v110.11.185.185.
Full textAbstract:
Abstract Follicular lymphoma (FL), one of the most common B cell non-Hodgkin’s lymphoma, is a germinal centre (GC)-derived malignancy, for which acquisition of the oncogenic t(14;18) translocation in the bone marrow constitute the genetic hallmark and early initiating event of FL pathogenesis. As t(14;18) is also present at low frequency in peripheral blood from healthy individuals (HI), it has been assumed that in HI, t(14;18) is carried by circulating quiescent naïve B-cells with restrained oncogenic potential. In sharp contrast, we recently demonstrated that in HI, t(14;18) is mainly carried by an expanding population of atypical B-cells presumably issued from the GC, displaying genotypic and phenotypic features of FL, and prone to constitute potent pre-malignant niches. Based on these data, we proposed that in HI, most t(14;18)+ cells were similarly rescued by BCL2 from apoptosis, and “frozen” at a differentiation stage where constitutive AID expression drives continuous somatic hypermutation (SHM) and class switch recombination (CSR) activity, two GC-associated mechanisms conferring a high propensity for further oncogenic aberrations. To test this model, we investigated the evolution of t(14;18)+ clones over time in HI by examining whether “FL-like” clonal development is associated with GC-specific processes. Using immunophenotypic, LR-PCR and mutation pattern analysis of the upstream switch μ flanking region from the translocated allele, we found that most circulating FL-like cells retain the GC-specific CD10 marker and carry highly mutated Sμ regions with a similar pattern to the mutated IgV genes, signing the maintenance of a GC-derived AID-mediated process. Moreover, as found for FL clones, the mutation load is higher in isotype switched than in sIgM+ t(14;18)+ cells, a difference maintained during the course of evolution (parallel acquisition of CSR/SHM on both functional and translocated alleles). We next analyzed intraclonal variation (ICV) as a way to determine how t(14;18)+ clones evolve over time based on their common BCL2/JH signature. In contrast to typical GC-derived memory B cells, which usually undergo transitory and extensive proliferation upon antigenic challenge without further SHM, we were able to construct genealogical trees for most circulating t(14;18)+ clones. Interestingly, few t(14;18)+ clones, although persistent and frequent, showed no clear evidence for clonal evolution but rather subclone selection. The presence of ICV and most importantly the identification of a somatically mutated common precursor through clonal arborescence confirm that FL-like cells not only display a GC-”frozen” phenotype but also provide direct support for the existence of premalignant niches from which cells undergo clonal evolution/selection and are constantly released. Although it remains currently unknown whether t(14;18)+ clones are directly issued from the GC founder or, similarly to FL, acquired the ability to invade other reactive GC for further rounds of SHM/CSR, ICV constitute an indirect signature for intense dynamics of the cells. Taken together, our results indicate that long-lived t(14;18)+ cells from HI are not conventional memory B-cells but recapitulate features of a “frozen” GC-derived population, able to undergo active AID-mediated processes while retaining at the same time dependence on BCR expression and presumably keeping the potential for intense trafficking between blood and tissues, a unique feature shared with FL cells.
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Lanasa, Mark C., Sallie D. Allgood, Susan L. Slager, Nicola Camp, Logan Spector, Laura Rassenti, Neil E. Kay, et al. "Family-Associated Monoclonal B Lymphocytosis Is Commonly Oligoclonal and Expresses Markers Associated with Adverse Risk in CLL." Blood 112, no. 11 (November 16, 2008): 3144. http://dx.doi.org/10.1182/blood.v112.11.3144.3144.
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Abstract Background and Significance : Chronic lymphocytic leukemia (CLL) is the most heritable hematologic malignancy; however, no common CLL predisposition genes are known. Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by small accumulations of B lymphocytes in the peripheral blood. MBL has a CLL-like immunophenotype, may progress to overt CLL, and is over represented in CLL families. Therefore, MBL observed in the context of familial CLL may be a marker of inherited risk for development of CLL. Detailed characterization of family-associated MBL may also provide mechanistic insights into the pathogenesis of familial CLL. Our strategy was to detail the biologic characteristics of CLL in family-associated MBL. Methods : Persons with MBL were identified by flow cytometric screening of peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. Flow cytometry was used to determine the surface immunophenotype including CD38 and intracellular ZAP-70. We defined MBL as populations of CD19+, CD5+, CD20lo, CD23+ B cells that comprised at least 2% of the CD19+ peripheral B cell compartment and did not exceed 5.0 × 109 MBL cells/L. RNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences. MBL cells were sorted in bulk for FISH for loci associated with clinical CLL. Results : Twenty-two out of 190 (12%) unaffected family members were found to have MBL. We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 32%; range 2%–97%). Nonetheless, the absolute size of the MBL clone was small, 15 of 17 individuals had < 200 × 106 MBL cells/L. CD38 expression (defined as CD38 surface expression in ≥30% of MBL cells) was observed in 8 of 18 subjects tested. ZAP-70 (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 4 of 12 participants. Among 12 subjects tested, 5 MBL expressed both surface IgD and IgM, 3 expressed IgD only, 2 expressed IgM only, and 2 did not express IgD or IgM. Analysis of immunoglobulin heavy and light chains has been completed in 8 individuals. Both immunoglobulin heavy chain variable (IgVH) region mutated (n = 12) and unmutated (n = 4) sequences were observed. Four of 8 individuals had 2 or more unrelated MBL clones (range 2–5), including one individual with both unmutated and mutated clones. Among the 16 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IgVH genes were 3–07 (3 MBL clones), 3–15 (3), and 4–34 (3). No VH1 family gene rearrangements were observed. In one individual, a VH3–07 MBL clone showed intraclonal diversification suggestive of antigen driven immunoglobulin sequence changes. Twenty productive light chain rearrangements were identified among the 16 MBL clones, with 11 Vλ and 9 Vκ genes used. We observed 6 productive rearrangements of Vλ1–51. MBL cells were bulk sorted for FISH from 9 subjects. Mono or biallelic deletion of 13q14.3 was observed in 5 subjects, the other 4 were normal. Conclusions : Our findings confirm that MBL is commonly observed among the unaffected family members from CLL kindreds. We found that some MBL clones express ZAP-70, CD38 or have unmutated IgVH genes and thus are similar to clinical CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. Small MBL clones are commonly oligoclonal. Importantly, although the immunoglobulin heavy and light chain genes rearranged in these MBL clones are all commonly used in CLL, the absence of VH1 and abundance of Vλ1–51 rearrangements do suggests differences in BCR usage between CLL and MBL. Further investigation of family associated MBL may clarify the genetics and immunobiology of familial CLL.
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Roulland, Sandrine, Nathalie Jouve, Agnes Bru, and Marie-Helene Delfau-Larue. "Somatic Mutation and Isotype Switch Events Outside Igv Gene Loci Provide New Evidence for a Role of Antigenic Challenge in Mantle Cell Lymphoma Etiology." Blood 126, no. 23 (December 3, 2015): 2647. http://dx.doi.org/10.1182/blood.v126.23.2647.2647.
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Abstract Mantle cell lymphoma (MCL) represents 5-10% of B-cell lymphomas and generally exhibits an aggressive clinical behavior. t(11;14) translocation, the genetic hallmark and early initiating event of MCL pathogenesis, lead to the constitutive CCND1 expression and consequently to cell-cycle deregulation. However, t(11;14) is also present in the blood from rare healthy individuals indicating that alone the t(11;14) is not sufficientfor malignant transformation. It has long been assumed in MCL that t(11;14)+ cells were actually carried by circulating naive and antigen-inexperienced B-cells. Yet, several reports, including long-term persistence of t(11;14)+ cells in lymphoma free-patients, biased immunoglobulin heavy variable chain gene (IgVH) repertoire and evidence of subset of cases with mutated IgVH genes led us to question this model and further investigate the role of antigenic challenge in MCL etiologic origin. As a first approach, we sought to determine the class-switch recombination (CSR) status of t(11;14)+ cells in MCL patients. CSR, as somatic hypermutation (SHM), are diversification mechanisms of the B cell receptor induced upon antigen encounter in the germinal center (GC) reaction, and absent in naive cells. Evidence for ongoing switch events in MCL has been previously reported with detection of mature switch transcripts, however in MCL cells, sIgM(D) expression remains clearly evident suggesting that effective deletional switch events occur infrequently on the functional allele. To characterize CSR in t(11;14)+ cells, we thus took advantage of their unique BCL1/IGH translocation signature assuming that BCL1/IGH fusions do not prevent CSR to occur on the downstream constant region of the IGH locus. Using 2 long-range PCR assays designed to amplify unswitched BCL1/Sµ and switched BCL1/Sγ regions, DNA from 30 MCL patients differing by their IgVH status were tested. Of the MCL cases, we confirmed that 7 of 30 (23%) were unmutated (UM, 100% germline homology) and 23 of 30 (77%) were mutated (M), considering as mutated all cases exhibiting any level of SHM (ranging from minimal: >0.3% to high rate: 7.0%). Although mutated BCRs were common in most MCL cases, we found that only 1 out of 30 MCL cases underwent CSR to IgG on the non-productive, translocated allele. Accordingly, this case carried a mutated IgVH gene with 97.5% germline identity in line with a GC/antigen-experienced signature of the tumor cells. Contrary to SHM events of the IgVH gene region, CSR deletional events appeared very infrequent in the bulk of MCL cells both on the functional and translocated allele, contrasting from findings in other GC-derived non-Hodgkin lymphomas where the same selective pressure to maintain a sIgM is accompanied by deletional switch events on the non-functional allele. To get more insights into a role of antigenic drive in MCL and AID targeting outside the VH gene loci, we next evaluated the clonal evolution of t(11;14)+ cells from 11 MCL cases (2 UM and 9 M) by analyzing SHM in the Sμ region on the translocated allele using sequencing of LR-PCR amplicons (with 2 subclones/patient). We found that 63% (7 out of 11) of BCL1/IGH clones display significant levels of SHM in the Sµ together with intra-Sµ indels (2/11) compatible with off-target AID-mediated events linked to chronic(?) antigen encounter. In addition, we found a good correlation between mutated BCR and the SHM levels in Sμ regions for a given patient. Importantly, in one instance subclones from a MCL case displayed intraclonal diversification (despite the fact that the expected rate of SHM is much lower in the Sµ region than in the IgVH region), indicative of an ongoing AID activity at least in a subset of MCL cases. In conclusion, we found that CSR events, although detectable in some MCL cases, do not appear to participate predominantly or to be selected during MCL lymphomagenesis. By contrast, analysis of SHM profiles both on the functional and the non-functional translocated allele provide evidence for an active AID activity in MCL cells inside and outside V-gene loci, highlighting the propensity of some t(11;14)+ clones to acquire a complementary oncogenic hit over time as a result of recurrent antigen challenge. Disclosures No relevant conflicts of interest to declare.
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Chen, Zhenghao, Gaspard Cretenet, Beatriz Valle-Argos, Francesco Forconi, Arnon P. Kater, Graham Packham, and Eric Eldering. "Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia." Blood 136, Supplement 1 (November 5, 2020): 36–37. http://dx.doi.org/10.1182/blood-2020-142839.
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Introduction. Altered metabolism is one of the hallmarks of cancer. CLL cells circulate between peripheral blood (PB) and lymph nodes (LN) which necessitates high metabolic plasticity. In LN, CLL cells receive proliferative and pro-survival signals from surrounding cells, and become metabolically activated. However, detailed insight into the altered metabolism of LN CLL and how this may be related to therapeutic responses is lacking. As it is technically difficult to obtain direct insight into CLL LN metabolism, we have applied a two-tiered strategy. By using PB samples taken from patients before/after treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR), which drives CLL cells out of the LN, combined with in vitro re-stimulation of TME signals, we indirectly mapped the metabolism of CLL in their TME, as well as the effects of IBR treatment. We hypothesized that the overlapping/distinct metabolites affected by IBR and in vitro stimulations would reflect the actual CLL metabolism in LN. Methods. PB samples were obtained from 7 CLL patients before or after 3 months of ibrutinib treatment. These paired samples were in vitro stimulated via CD40 and B cell receptor (BCR), which are potential key signals within the tumour microenvironment (TME). Seahorse extracellular flux (ECF) analyses, expression of activation markers (CD95, pS6 by FACS), RNA was isolated for expression of Myc (major driver of metabolic reprogramming) and its target genes, and metabolomics by mass-spec was performed. Results. ECF analyses showed that in comparison to BCR stimulated PB CLL cells, stimulation by CD40 resulted in a high increase of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). A prominent effect on OXPHOS and glycolytic activity was confirmed in direct LN samples, and indirectly by marker analyses in LN emigrants using CXCR4/CD5 staining [1]. Subsequent metabolomics analyses showed that metabolic reprogramming following CD40 or BCR stimulation revealed both shared and distinct responses. The affected metabolic pathways, predicted by significantly changed metabolites, were compared in a pairwise fashion; upregulated by CD40 and BCR but downregulated by IBR, respectively. The results demonstrated 5 upregulated pre-defined pathways (KEGG) by both CD40 and BCR triggering: purine metabolism, Warburg effect, lysine degradation, glucose-alanine cycle and glutamate metabolism. In contrast, the following pathways indicated the two signals had distinct functions on regulating metabolism: CD40 signalling mostly regulates amino acid metabolism, tricarboxylic acid cycle (TCA) and mitochondrial metabolism related to oxidative phosphorylation (OXPHOS) and energy production. BCR signalling mainly involves glucose and glycerol metabolism, which are usually related to biosynthesis. CLL cells from IBR-treated patients showed enhanced BCR responsiveness, in line with the increased in surface IgM expression upon IBR [2]. In contrast, IBR treatment suppressed in vitro CD40 activation, which was accompanied by a lower CD40 expression. Metabolomics analyses also demonstrated that CD40 responses decreased but BCR response increased after IBR. Additionally, analyses of Myc and its target genes showed that they are induced after BCR as well as CD40 stimulation. Effects of IBR on Myc (target) expression were variable for BCR and reduced for CD40 stimulation. Conclusions. In vivo IBR treatment suppresses CD40 expression and activation and enhances BCR responsiveness. Metabolic changes of CLL in LN are recapitulated by these two signals, while IBR treatment shows opposite effects, together providing indirect insight into the LN metabolism. In LN, CD40 may play a prominent role to enhance most of the key metabolic pathways, particularly OXPHOS. This is the first study to describe the metabolic network of CLL cells in LN, and the long-term effects of IBR may yield new clues to therapy response and resistance. References 1. Calissano, Carlo, et al. "Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells." Molecular Medicine 17.11 (2011): 1374-1382. 2. Drennan, Samantha, et al. "Ibrutinib therapy releases leukemic surface IgM from antigen drive in chronic lymphocytic leukemia patients." Clinical Cancer Research 25.8 (2019): 2503-2512. Disclosures Forconi: AbbVie: Honoraria, Other: Fees for cosulting or advisory role, received travel and expenses, Speakers Bureau; Janssen: Honoraria, Other: Fees for cosulting or advisory role, received travel and expenses, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Menarini: Other: Fees for cosulting or advisory role; Astra Zeneca: Other: Fees for cosulting or advisory role; Gilead: Research Funding. Kater:Roche: Research Funding; Abbvie: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Janssen: Research Funding. Eldering:Janssen: Research Funding; Celgene: Research Funding; Genentech: Research Funding.
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Damle, Rajendra N., Sonal Temburni, Cristina Sison, Jaison Jain, Jacqueline C. Barrientos, Jonathan E. Kolitz, Kanti R. Rai, and Nicholas Chiorazzi. "Reciprocal Densities of CXCR4 and CD5 Define Subfractions of Chronic Lymphocytic Leukemia Clones Differing in Phenotype and Response to Environmental Stimuli: Towards a Better Definition of Targetable Components of Leukemic Clones." Blood 124, no. 21 (December 6, 2014): 3322. http://dx.doi.org/10.1182/blood.v124.21.3322.3322.
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Abstract Although chronic lymphocytic leukemia (CLL) is an accumulative disorder of CD5+ B cells, a proliferative fraction exists and the extent of this proliferation correlates with clinical course. We previously demonstrated heterogeneous in vivo labeling kinetics of cells in 3 clonal fractions sorted based on reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and CD5 from CLL cases recruited on an in vivo labeling study. In that study, the CXCR4dimCD5br fraction contained more 2H-labeled DNA and hence recently divided cells (proliferative fraction) than the CXCR4brCD5dim(resting) fraction. Here, we have analyzed multiple CXCR4/CD5 based subclonal fractions and characterized their phenotypic differences and ability to respond to signals via the BCR and CXCR4 or a combination of the two. PBMCs from a cohort of 50 untreated IgM+ CLL cases were used for this study. Subclonal fractions marked based on differences in relative densities of CXCR4 and CD5 by CLL cells are referred to as CXCR4br, CXCR4int or CXCR4dim and CD5br, CD5int or CD5dim. Sub-populations adjacent to each other on flow cytometric plots differed at least 5 fold in the densities of both CXCR4 and CD5. When subcategorizing CLL clones based on these markers, 9 subfractions were identified. To permit comparisons between these fractions, each was defined as containing only 2-5% of the total clonal CD19+CD5+population while ensuring no overlap in CXCR4 or CD5 densities among each fraction. Interestingly, certain phenotypic and signaling features consistently clustered with distinct subfractions of the CLL clone. Subfraction analyses confirmed an enrichment of Ki-67+ cells in the CXCR4dimCD5br proliferative fraction of every CLL case. In addition, this subfraction showed the highest percentage of cells expressing smIgM and smIgD (p<0.01). Relative density (RD) of expression was deduced as a ratio of mean fluorescence intensity (MFI) of a molecule expressed by any subfraction compared to that expressed by CXCR4intCD5int cells within the same clone. When considering all cases the CXCR4dim CD5dim cells showed the highest RD of smIgM. However, among U-CLL cases the CXCR4dim CD5br cells expressed the highest RD for smIgM, and the CXCR4dim CD5dim subfraction showed the highest RD of smIgM expression in M-CLLs. Overall, both CXCR4dim CD5br and CXCR4int CD5br showed the highest RD for IgD expression, whereas among U-CLL and M-CLL cases the CXCR4int CD5br and CXCR4dim CD5int subfractions showed the highest RD of smIgD expression respectively. The CXCR4brCD5br subfraction was characterized by the highest % and RD of CD79b, the BCR signaling molecule, and CD22 and CTLA-4, negative regulators of signaling. However, Siglec-10, another negative regulator of BCR signaling, was most highly expressed both in % and RD by the CXCR4dim CD5br subfraction in all CLL cases. Finally, the highest percentages of cells expressing CD21, CD38, CD43, CD69, CD180 associated best with the 3 CD5br subfractions. Of note, CD38 was expressed at highest RD in the CXCR4dim CD5dim in M-CLL cases but in the CXCR4dim CD5brsubfraction in the U-CLL cases. When analyzing phosphorylation of Akt, Erk, p38MAPK and Syk by phosphoflow among all cases, the CXCR4brCD5br fraction exhibited the highest constitutive levels. Constitutive levels of phospho-Btk and - Syk were significantly higher in the CXCR4int CD5br subfraction in M-CLL than in U-CLL cases (p<0.05). Anti-BCR mAbs induced significant increases in phospho-p38MAPK and -Akt in the CXCR4br CD5br subfraction when comparing U-CLL to M-CLL cells. CXCL12 induced significant increases in phospho- Akt and -STAT-5 in CXCR4int CD5br subfraction in U-CLL cases (p<0.01), whereas a combination of signals via BCR and CXCR4 induced increases in phospho-Erk in the CXCR4int CD5br and CXCR4br CD5brpopulations in M-CLL cases. Together, these findings highlight the impact of the molecules expressed by the different subfractions on their ability to relay/inhibit signals elicited via the BCR or CXCR4. They also provide a general frame of reference to further understand functional post-replicative intraclonal heterogeneity of CLL clones and help define aggressive subfractions of the CLL clone as target populations for future therapies. Disclosures No relevant conflicts of interest to declare.
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Macana, Yesid Alejandro Mariño, and Miguel Alonso Rodríguez Melo. "El punto de marchitez permanente (PMP) en melina (Gmelina arborea L. Roxb) para la Costa Caribe colombiana ¿una característica para la selección de clones?" Corpoica Ciencia y Tecnología Agropecuaria 11, no. 2 (November 29, 2010): 116. http://dx.doi.org/10.21930/rcta.vol11_num2_art:201.
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<p>Gmelina arborea (melina), especie forestal introducida en Colombia, se planta en áreas de la costa norte de Colombia caracterizado por presentar relativamente largas estaciones secas con baja disponibilidad de agua edáfica, principal limitante de su productividad. Con el fin de asignar los mejores materiales de siembra a ofertas ecofisiológicas específicas, se ha avanzado en su mejoramiento genético, confirmando si sus respuestas fisiológicas al estrés hídrico sirven como parámetro de selección de clones tolerantes a la baja disponibilidad de agua. Este estudio, desarrollado bajo condiciones de invernadero en el municipio de Zambrano (Bolivar), evaluó la respuesta de 27 clones de G. arborea a la tensión de humedad del suelo, utilizando la capacidad de campo (CC) y el punto de marchitez permanente (PMP) como criterio para la asignación de clones a sitios potenciales de plantación. Los 27 clones pertenecientes a la colección Pizano SA fueron sembrados en bloques cilíndricos de suelo no perturbado y extraídos de nueve puntos pertenecientes a diferentes unidades cartográficas de las series de suelos en la región de Zambrano. Se encontró que el PMP varió entre -4,8 y -9 bar. En 17 clones los valores del PMP se expresaron independientemente de las propiedades fisicoquímicas de los suelos, mientras que en los 10 restantes, los valores del PMP del suelo parecen asociarse con sus propiedades fisicoquímicas. Para esta última respuesta el análisis de los componentes de varianza indica que en cinco de los diez clones presenta variación intraclonal atribuida a la topófisis. Los resultados confirman la posibilidad de emplear el PMP como criterio para la asignación de clones a sitios potenciales para plantaciones comerciales de melina.</p><p> </p><p> </p><p><strong>The permanent wilting point (PWP) in yemane (Gmelina arborea L. Roxb) for the Colombian Caribbean Coast ¿a feature for the selection of clones?</strong></p><p>Gmelina arborea (melina), an introduced tree species in Colombia, is planted in areas of the north coast of Colombia which is characterized by long, dry seasons with low soil water availability, limiting primary productivity. In order to assign the best planting materials to specific ecophysiological conditions, progress has been made in genetic improvement, confirming whether the physiological responses to water stress are an appropriate parameter to select clones tolerant to the low water availability. This study was conducted under greenhouse conditions in the municipality of Zambrano (Bolivar), and evaluated the response of 27 clones of G. Arborea to soil moisture tension, using field capacity (FC) and permanent wilting point (PWP) as criteria for the assignment of clones to potential planting sites. The 27 clones, from the Pizano SA collection, were planted in cylindrical blocks of undisturbed soil at nine points taken from different units of the soil series mapping of the Zambrano region. PWP was found to range between -4.8 and -9.0 bar. In 17 clones, PWP values were not affected by the physicochemical properties of the soil, while the remaining 10 PWP values appeared to be associated with these properties. Of these 10, an analysis of the variance components indicates that five of the ten clones show intraclonal variation attributed to topophysis. The results confirm the possibility of using PWP as a criterion for assigning clones to potential sites for the commercial cultivation of melina.</p><p> </p>
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Jeusset, Lucile, Nika Abdollahi, Thibaud Verny, Marine Armand, Anne Langlois De Septenville, Frédéric Davi, and Juliana Silva Bernardes. "ViCloD, an interactive web tool for visualizing B cell repertoires and analyzing intraclonal diversities: application to human B-cell tumors." NAR Genomics and Bioinformatics 5, no. 2 (March 29, 2023). http://dx.doi.org/10.1093/nargab/lqad064.
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Abstract High throughput sequencing of adaptive immune receptor repertoire (AIRR-seq) has provided numerous human immunoglobulin (IG) sequences allowing specific B cell receptor (BCR) studies such as the antigen-driven evolution of antibodies (soluble forms of the membrane-bound IG part of the BCR). AIRR-seq data allows researchers to examine intraclonal differences caused primarily by somatic hypermutations in IG genes and affinity maturation. Exploring this essential adaptive immunity process could help elucidate the generation of antibodies with high affinity or broadly neutralizing activities. Retracing their evolutionary history could also clarify how vaccines or pathogen exposition drive the humoral immune response, and unravel the clonal architecture of B cell tumors. Computational methods are necessary for large-scale analysis of AIRR-seq properties. However, there is no efficient and interactive tool for analyzing intraclonal diversity, permitting users to explore adaptive immune receptor repertoires in biological and clinical applications. Here we present ViCloD, a web server for large-scale visual analysis of repertoire clonality and intraclonal diversity. ViCloD uses preprocessed data in the format defined by the Adaptive Immune Receptor Repertoire (AIRR) Community. Then, it performs clonal grouping and evolutionary analyses, producing a collection of useful plots for clonal lineage inspection. The web server presents diverse functionalities, including repertoire navigation, clonal abundance analysis, and intraclonal evolutionary tree reconstruction. Users can download the analyzed data in different table formats and save the generated plots as images. ViCloD is a simple, versatile, and user-friendly tool that can help researchers and clinicians to analyze B cell intraclonal diversity. Moreover, its pipeline is optimized to process hundreds of thousands of sequences within a few minutes, allowing an efficient investigation of large and complex repertoires.
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Gemenetzi, Katerina, Fotis Psomopoulos, Alejandra Carriles, Maria Gounari, Claudia Minici, Karla Plevova, Lesley A. Sutton, et al. "HIGHER ORDER IMMUNOGLOBULIN REPERTOIRE RESTRICTIONS IN CLL: THE ILLUSTRATIVE CASE OF STEREOTYPED SUBSETS #2 AND #169." Blood, October 9, 2020. http://dx.doi.org/10.1182/blood.2020005216.
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Chronic lymphocytic leukemia (CLL) major stereotyped subset #2 (IGHV3-21/IGLV3-21, ~2.5% of all CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset #169 (IGHV3-48/IGLV3-21, ~0.2% of all CLL) is related to subset #2 as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B cell receptor immunoglobulin (BcR IG). Branching evolution of the predominant clonotype through intraclonal diversification (ID) in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the two subsets were highlighted by the finding of shared SHMs within both the heavy and the light chain genes in all analyzed cases at either clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for IG homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset #169 IG retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset #2, including the SHM at the linker region, and from a molecular standpoint belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets #2 and #169 are very closely related, displaying shared IG features that can be explained only in the context of shared functional selection.
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Bagnara, Davide, Catherine Tang, Jennifer R. Brown, Siddha Kasar, Stacey Fernandes, Monica Colombo, Stefano Vergani, et al. "Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course." Frontiers in Oncology 11 (May 25, 2021). http://dx.doi.org/10.3389/fonc.2021.640731.
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Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.