Academic literature on the topic 'BCR intraclonal analysis'

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Le séquençage de nouvelle génération a permis aux chercheurs de réaliser des analyses approfondies du paysage du répertoire immunologique. Cependant, une préoccupation importante dans ces études est le coût informatique de l'analyse de millions de séquences avec une complexité, une variabilité et une capacité de mutation inhérentes, imposant des défis informatiques et nécessitant le développement de méthodes efficaces. Ce défi est encore plus évident dans le contexte clinique qui n'a pas nécessairement accès à des professionnels ayant des compétences informatiques ou des ressources informatiques robustes. Ainsi, l'objectif principal de cette thèse est de développer un ensemble d'outils dédiés qui seront utilisés dans l'environnement clinique, pour le diagnostic médical et les soins aux patients, et dans l'environnement de recherche, pour effectuer une analyse approfondie et à grande échelle du répertoire. Nous avons conçu et implémenté de multiples algorithmes et les avons rassemblés dans un pipeline interactif de visualisation du répertoire BCR afin de faciliter le processus d'intégration de l'analyse du répertoire BCR dans les pratiques médicales
Next-generation sequencing has enabled researchers to conduct in-depth analyses of the immunological repertoire landscape. However, a significant concern in these studies is the computational cost of analyzing millions of sequences with inherent complexity, variability, and mutational capacity, imposing computational challenges and necessitating the development of efficient methods. This challenge is even more evident in the clinical context that does not necessarily have access to professionals with computing skills or robust computational resources. Thus, the main goal of this thesis is to develop a set of dedicated and integrated tools to be used in the clinical environment, for medical diagnostic and patient care, and in the research environment, to perform large-scale and in-depth repertoire analysis. We have designed and implemented multiple algorithms and gathered them in a user-friendly interactive BCR repertoire visualization pipeline to facilitate the process of integrating BCR repertoire analysis into medical practices