Ongoing Somatic Hypermutation Research Articles

B-cell characteristics define HCV reinfection outcome

B-cell characteristics define HCV reinfection outcome

Analysis of immunoglobulin/T-cell receptor repertoires by high-throughput RNA sequencing reveals a continuous dynamic of positive clonal selection in follicular lymphoma.

Follicular lymphoma (FL) course is highly variable, making its clinical management challenging. In this incurable and recurring pathology, the interval between relapses tends to decrease while aggressiveness increases, sometimes resulting in the transformation to higher-grade lymphoma. These evolutions are particularly difficult to anticipate, resulting from complex clonal evolutions where multiple subclones compete and thrive due to their capacity to proliferate and resist therapies. Here, to apprehend further these processes, we used a high-throughput RNA sequencing approach to address simultaneously the B-cell immunoglobulin repertoires and T-cell immunoglobulin repertoires repertoires of lymphoma cells and their lymphoid microenvironment in a large cohort of 131 FL1/2-3A patients. Our data confirm the existence of a high degree of intra-clonal heterogeneity in this pathology, resulting from ongoing somatic hyper-mutation and class switch recombination. Through the evaluation of the Simpson ecological-diversity index, we show that the contribution of the cancerous cells increases during the course of the disease to the detriment of the reactive compartment, a phenomenon accompanied by a concomitant decrease in the diversity of the tumoral population. Clonal evolution in FL thus contrasts with many tumors, where clonal heterogeneity steadily increases over time and participates in treatment evasion. In this pathology, the selection of lymphoma subclones with proliferative advantages progressively outweighs clonal diversification, ultimately leading in extreme cases to transformation to high-grade lymphoma resulting from the rapid emergence of homogeneous subpopulations.

Somatic hypermutation introduces bystander mutations that prepare SARS-CoV-2 antibodies for emerging variants

Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2-neutralizing antibodies (nAbs). However, several potent SARS-CoV-2 antibodies carry no or only a few mutations, leaving the question of how ongoing SHM affects neutralization unclear. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, for some antibodies, including antibodies of the public clonotype VH1-58, neutralization of Wu01 remained unaffected. Although mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical for Omicron BA.1/BA.2 neutralization. We exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2 neutralizer. These findings broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation but also contributes to antibody diversification, thus increasing the chances of neutralizing viral escape variants.

Detection of Occurrent Somatic Hypermutation and Associated Gene Expression Profile in Single B Cells

Introduction Somatic Hypermutation (SHM) is an endogenous mutator mechanism in B lymphocytes that preferentially targets the immunoglobulin V(D)J coding region and facilitates antibody affinity maturation. The exact frequency and impact of SHM in individual B cells remains elusive, as previous quantifications only considered cumulative outcomes at population level. We therefore employed single-cell RNA sequencing on primary follicular lymphoma (FL) cells, a B-cell malignancy retaining the germinal center (GC) B-cell phenotype, to identify and quantify individual SHM events. This approach allowed us to detect neovariants, defined as single nucleotide differences that originate from recent SHM events, and to explore the gene expression profiles of the B lymphoma cells with neovariants, particularly focusing on genes and pathways effectuating SHM. Methods We collected and processed samples from 12 follicular lymphoma (FL) and five chronic lymphocytic leukemia (CLL) patients as non-germinal center controls. Single cell whole cDNA libraries were prepared by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with semi-nested amplification using 3′ constant domain primers followed by 10X Genomics prep. Both single cell libraries were sequenced in paired-end mode on Illumina HiSeq 4000. We developed a customized method to preprocess scBCR data designed specifically for highly mutated FL BCR sequences. To ensure data quality and validity of putative neovariants, we applied a comprehensive filtering system, including stringent thresholds in terms of numbers of individual immunoglobulin (IG) mRNA molecules sequenced as defined by their UMI, and reads per position obtained from the median of the estimator's distribution, base quality scores, exclusion of cell doublets, position within the V(D)J region, and proportion of cell-level variants. Based on a reported IG mRNA half-life of 3 h and assuming a first-order decay, the filtering criteria implied that detected neovariants were created between 1 and 7 hours prior to the time of sampling. Results A median of 1112 cells (range: 215-16432) per case were analyzed. 1239 IG neovariants were detected in a total of 334 FL cells in 7 of the 12 FL cases. Neovariants were undetectable in control CLL cells. A remarkably high proportion (95,4%) of neovariants were found within known AID motifs (WRCY, WA, and RCG), suggesting active AID involvement in the hypermutation process. The observed fractions of FL cells carrying recent SHM events indicated initiation of FL pathophysiology at least 11,2 months prior to sampling. To further evaluate the role of AID, we compared gene expression profiles between FL cells with and without neovariants. The results demonstrated not only higher expression of AID but as well of pathways involved in SHM. Mismatch repair (hsa03430, 9 enriched genes with p.adjust < 0.001) and base excision repair (hsa03410, 17 enriched genes with p.adjust = 0.01), together with DNA replication (hsa03030, 14 enriched genes with p.adjust < 0.001), were the three most prominent pathways with upregulated genes. In cells with detectable neovariants, the MLH1/MSH2 genes, associated with the recognition of the deamination process, as well as POLH gene, which is involved in error-prone mutations in MMR, were highly expressed. Overall, we observed a more prominent expression of genes related to MMR than BER in cells with neovariants supported by a higher normalized enrichment score (NES) in MMR (MMR:1.93 and BER:1.6). This suggests not only an active SHM process but also potential interplay between AID and MMR in driving SHM in FL. Conclusions Here we report for the first time quantitative detection of ongoing somatic hypermutation at single cell level in FL. Based on the lifespan of IG mRNA, simultaneous detection of two IG mRNA transcripts differing by one (or a few) nucleotides only within a single cell permits to estimate the timing when this SHM event occurred. The detection of this phenomenon in 58% of FL samples and its association with AID expression suggests that AID-induced mutagenesis might be acting at a much higher rate than expected. Quantification of SHM at the single-cell level should facilitate the understanding of the cellular heterogeneity and dynamics in physiological GC reactions but also may offer novel insights into the AID-dependent shaping of the genomic landscape of germinal center B-cell neoplasms.

An Immunogenetic Imprint of Ongoing Antigen Interactions in Hairy Cell Leukemia Expressing IGHV4-34 Receptors

Hairy cell leukemia (HCL) is a rare indolent B-cell malignancy thought to derive from a BRAF-mutant antigen-experienced B cell. Supporting evidence includes a transcriptional profile that is most similar to post-germinal center (GC) memory B cells; the presence of somatic hypermutation (SHM) in the majority of HCL cases; and, the preferential usage of the IGHV4-34 gene in the minority of classic HCL (HCLc) cases with low or no SHM. IGHV4-34-expressing HCLc is clinically more aggressive than other HCLc; moreover, it is enriched for BRAF-wildtype cases while it displays frequent MAP2K1 mutations. Overall, this constellation of features raises questions regarding the precise nature and ontogeny of IGHV4-34-expressing HCLc. Here we addressed this issue through investigating the subclonal architecture of the B cell receptor immunoglobulin (BcR IG) gene repertoire in a series of 17 cases with a typical phenotype of HCLc expressing the IGHV4-34 gene. Previous Sanger analysis had documented 10 cases carrying IGHV genes with 100% germline identity (GI), 2 cases with minimal SHM (GI>99%) and 5 with a more significant SHM load (GI<98%). Total RNA was isolated from peripheral blood mononuclear cells and IGHV-IGHD-IGHJ rearrangements were RT-PCR amplified and subjected to NGS sequencing using a paired-end protocol. Quality filtering of NGS raw reads was performed with a purpose-built bioinformatics pipeline and downstream analysis was performed with the IMGT/HighV-QUEST, tripr and IgIDivA softwares. Clonotypes were defined as clusters of rearrangement sequences expressing the IGHV4-34 gene and bearing identical variable heavy complementarity-determining region 3 (VH CDR3) amino acid (aa) sequences. Variants of a given clonotype i.e. gene rearrangement sequences with a VH CDR3 of identical length differing in up to 2 aa positions were defined as sub-clonotypes; these are likely to have differentiated from the main clonotype due to ongoing SHM. For the analysis of intraclonal diversification (ID) within the clonotypic rarranged IGHV genes, we used the metrics 'convergence score', describing the tendency of the BcR IG to acquire more mutations, and 'maximal pathway length', showing whether ongoing SHM acquisition takes place. Overall, we obtained 3,238,715 raw reads (median 178,446 reads/sample). Of these, 2,226,444 reads corresponded to productive IGHV4-34 gene rearrangements (median 127,647 sequences/sample) that were assigned to a total of 10,532 clonotypes (median 593 clonotypes/sample). The median frequency of the dominant clonotype was 64.4%, indicating a significant subclonal branching due to ID. Indeed, overall, we detected 5,267 subclonotypes (median 213 subclonotypes/sample) corresponding to 242,271 unique nucleotide (nt) variants (median 8098 nt variants/sample). ID was observed in both IG-mutated (<100% GI) and IG-unmutated (100% GI) cases, albeit was significantly (p=0.01) more extensive in the former (median number of nucleotide variants/sample: 21,306 vs 7,099, respectively). Next, we compared the present HCLc dataset against a corresponding dataset produced following the same approach from 11 IG-mutated CLL (M-CLL) cases expressing the IGHV4-34 gene, of which 5 belonged to stereotyped subset #4, notable for a high degree of ID. No statistically significant differences were identified in convergence scores between HCLc vs CLL, indicating that the former also displays considerable ID despite an overall lower number of clonal SHMs (underlying the higher GI at the clonal level i.e. by Sanger sequencing analysis). Moreover, HCLc exhibited significantly (p=0.002) higher maximal pathway lengths compared to CLL, suggesting that acquisition of subclonal SHM in IGHV4-34 expressing HCLc is an ongoing process that takes place progressively in many steps. Also relevant to mention, different recurrent replacement SHMs were seen in IGHV4-34 expressing HCLc vs CLL: indicatively, G36S in VH CDR1 was present in subclones of 11/17 HCLc cases whereas it was totally absent in CLL. In conclusion, we report a complex immunogenetic architecture for IGHV4-34 expressing HCLc with distinctive, disease-biased features and extensive ID, likely mediated by ongoing antigen interactions. This finding questions a post-GC origin for at least a subgroup of HCLc, while also highlighting the need to reappraise the ontogenetic relationship of this particular HCLc subgroup vs all other HCLc.

Prevalence of Acquired N-Glycosylation Sites at the Single Cell Level in Follicular Lymphoma

Follicular lymphoma (FL), a malignancy derived from germinal center B cells, is characterized by ongoing somatic hypermutation of the immunoglobulin variable region (IGV) genes. This somatic hypermutation can introduce novel N-glycosylation sites (N-sites), consisting of Asn-X-Ser/Thr motifs, in the IGV resulting in the addition of glycans at these sites. Binding of lectins, such as DC-SIGN, to these glycosylated surface immunoglobulins (i.e., B cell receptors) activates the B cell receptor pathway in an antigen-independent manner, promoting FL B cell survival and proliferation. Previous studies have concluded that the acquisition of N-sites is a stable and clonal event during FL evolution (Odabashian et al., Blood 2020). Here, we investigated the dynamics and potential impact of N-site prevalence at a single cell level, in paired heavy and light chains, across sites and over time in 16 patients with FL. We profiled tumor fine needle aspirates from patients enrolled in two clinical trials (NCT02927964, NCT03410901). 15 of 16 patients were previously, but not recently treated, while one was previously untreated. Samples were collected from different tumor sites before treatment, on-treatment, and at tumor progression. Single cell suspensions were subjected to single cell B cell receptor (scBCR) and RNA (scRNA) sequencing using the 10X chromium platform. Only BCR sequences belonging to patient-specific dominant malignant heavy and light chain clones were considered for further analysis. In total, we analyzed more than 250,000 FL tumor cells from 89 tumor samples from 16 FL patients. While most patients acquired at least one N-site as an early and stable event that was conserved across distinct tumor sites and over time, 3 patient cases proved to be exceptions: one patient had no detectable N-sites in either the heavy or light chain IGV genes at any tumor site or timepoint. In a second patient, only one of two tumor sites exhibited a novel N-site, and this was probably acquired after divergence of the tumor populations to distinct tumor sites (Haebe, Shree et al., Blood 2021). Interestingly, clinical tumor progression in this patient arose from selection and expansion of an N-site negative subclone, despite the putative survival advantage conferred by acquired N-sites. In the third patient, N-sites were acquired in a precursor cell before subclonal divergence, but subsequently overtaken by an N-site-negative subpopulation, which comprised up to 90% of tumor cells at a given site (Figure 1A). These results suggest that other acquired features can out-compete the selective advantage of N-glycosylation. To elucidate biological differences between cells with and without N-sites within each sample, we integrated scBCR and scRNA data for the third patient using shared cell barcodes. N-site positive cells exhibited a higher expression of the BCR pathway and inflammatory response, while cells without N-sites had higher activity of metabolic processes, including oxidative phosphorylation and glycolysis (Figure 1B). In conclusion, while acquired N-sites likely support FL pathogenesis through antigen-independent BCR signaling in many FL patients, selection and expansion of N-site negative cells in some patients in our study suggests that N-site negative cells can thrive, and likely depend on other pathways, with our preliminary results pointing to differential metabolic regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IgIDivA: immunoglobulin intraclonal diversification analysis.

Intraclonal diversification (ID) within the immunoglobulin (IG) genes expressed by B cell clones arises due to ongoing somatic hypermutation (SHM) in a context of continuous interactions with antigen(s). Defining the nature and order of appearance of SHMs in the IG genes can assist in improved understanding of the ID process, shedding light into the ontogeny and evolution of B cell clones in health and disease. Such endeavor is empowered thanks to the introduction of high-throughput sequencing in the study of IG gene repertoires. However, few existing tools allow the identification, quantification and characterization of SHMs related to ID, all of which have limitations in their analysis, highlighting the need for developing a purpose-built tool for the comprehensive analysis of the ID process. In this work, we present the immunoglobulin intraclonal diversification analysis (IgIDivA) tool, a novel methodology for the in-depth qualitative and quantitative analysis of the ID process from high-throughput sequencing data. IgIDivA identifies and characterizes SHMs that occur within the variable domain of the rearranged IG genes and studies in detail the connections between identified SHMs, establishing mutational pathways. Moreover, it combines established and new graph-based metrics for the objective determination of ID level, combined with statistical analysis for the comparison of ID level features for different groups of samples. Of importance, IgIDivA also provides detailed visualizations of ID through the generation of purpose-built graph networks. Beyond the method design, IgIDivA has been also implemented as an R Shiny web application. IgIDivA is freely available at https://bio.tools/igidiva

High-throughput sequencing of IgH gene in minor salivary glands from Sjögren's syndrome patients reveals dynamic B cell recirculation between ectopic lymphoid structures.

B cells play a central role in Sjögren's syndrome (SS) whereby autoreactive B-cells populate ectopic germinal centres (GC) in SS salivary glands (SG) and undergo somatic hypermutation (SHM) and class-switch recombination of the immunoglobulin genes. However, the capacity of specific B cell clones to seed ectopic GC in different SG and undergo clonal diversification is unclear. To unravel the dynamics of B cell recirculation among minor SG biopsies, we investigated the immunoglobulin heavy chain (IgH) gene usage and the pattern of SHM using a high-throughput sequencing approach. We generated ~166,000 reads longer than 350bp and detected 1631 clonotypes across eight samples from four different SS patients, all characterised by the presence of functional ectopic GC as demonstrated by the expression of activation-induced cytidine deaminase. A large number of shared clonotypes were observed among paired mSG biopsies from each patient but not across different patients. Lineage tree analysis revealed significant clonal expansion within the mSG with the identification of shared dominant B cell clones suggestive of extensive recirculation across different SG. Several shared clonotypes with high proliferating capacity displayed IgH-VH gene usage common in autoreactive B cells, including VH1-69, which is typical of rheumatoid factor+ B cells representing potential lymphoma precursors. The complex dynamic recirculation of B cells that we observed within ectopic GC responses linked with their ability to independently proliferate, undergo ongoing SHM and Ig class-switching within individual glands may explain the difficulty in achieving consistent eradication of ectopic GCs following B cell depleting agents reported in different studies.

Impact of a Faulty Germinal Center Reaction on the Pathogenesis of Primary Diffuse Large B Cell Lymphoma of the Central Nervous System

Simple SummaryThe pathogenetic mechanisms and peculiar tropism of primary CNS lymphoma (PCNSL) of the central nervous system (CNS) have been the subject of debate for decades. Hypothesis-driven targeted molecular studies have revealed that PCNSLs derived from self-/polyreactive B cells that have escaped developmental control mechanisms. The early acquisition of activating mutations targeting the B cell receptor pathway provides a survival advantage. The failure of the germinal center (GC) reaction and its checkpoints increases tumor B cell affinity for the CNS. During this faulty GC reaction, PCNSL tumor cells acquire further oncogenic alterations converging on the Toll-like receptor, B cell receptor, and NF-κB pathway. These activated pathways sustain proliferation. Concomitantly, cells become unable to complete terminal B cell differentiation, becoming trapped within the vicious cycle of the GC reaction as low-affinity IgM+ B cells related to memory cells.Primary lymphoma of the central nervous system (PCNSL, CNS) is a specific diffuse large B cell lymphoma (DLBCL) entity confined to the CNS. Key to its pathogenesis is a failure of B cell differentiation and a lack of appropriate control at differentiation stages before entrance and within the germinal center (GC). Self-/polyreactive B cells rescued from apoptosis by MYD88 and/or CD79B mutations accumulate a high load of somatic mutations in their rearranged immunoglobulin (IG) genes, with ongoing somatic hypermutation (SHM). Furthermore, the targeting of oncogenes by aberrant SHM (e.g., PIM1, PAX5, RHOH, MYC, BTG2, KLHL14, SUSD2), translocations of the IG and BCL6 genes, and genomic instability (e.g., gains of 18q21; losses of 9p21, 8q12, 6q21) occur in these cells in the course of their malignant transformation. Activated Toll-like receptor, B cell receptor (BCR), and NF-κB signaling pathways foster lymphoma cell proliferation. Hence, tumor cells are arrested in a late B cell differentiation stage, corresponding to late GC exit B cells, which are genetically related to IgM+ memory cells. Paradoxically, the GC reaction increases self-/polyreactivity, yielding increased tumor BCR reactivity for multiple CNS proteins, which likely contributes to CNS tropism of the lymphoma. The loss of MHC class I antigen expression supports tumor cell immune escape. Thus, specific and unique interactions of the tumor cells with resident CNS cells determine the hallmarks of PCNSL.

“Snapshotting” Somatic Hypermutation in Single Follicular Lymphoma Cells

IntroductionFollicular Lymphoma (FL) is one of the most prevalent B-cell neoplasms and despite recent advances remains incurable in most cases. FL cells are malignant counterparts of normal germinal center B-cells. At molecular level FL are characterized by the t(14;18) which results in the overexpression of the BCL-2. These apoptosis resistant cells accumulate somatic mutations associated with tumorigenesis and progression. Whereas several mutagenic mechanisms shape the FL genomic landscape, up to 22% of the mutations may be attributed to activation-induced cytidine deaminase (AID) activity.Under physiological conditions AID is responsible for somatic hypermutation (SHM) in immunoglobulin genes (IG) of germinal center B-cells. In fact, it is accepted that ongoing SHM still occurs at relatively high rates in FL cells as compared with other germinal center lymphoid neoplasms. Moreover, we recently demonstrated the direct effect of AID overexpression inducing somatic mutations driving murine and human B-cell neoplasm progression in vivo. Therefore, unveiling the molecular basis of AID activity in lymphoma cells remains essential to understand lymphomagenesis and to develop novel targeted therapies.The advent of single-cell high-throughput sequencing has enabled the analysis of molecular events at an unprecedented resolution. Therefore, we designed a study to capture AID-induced somatic hypermutation at the single cell level in FL.MethodsTumor samples derived from 14 FL patients were analyzed, as controls, we chose a B-cell malignancy with lower SHM rates and included 5 samples derived from chronic lymphocytic leukemia (CLL) and 2 from monoclonal B lymphocytosis (MBL). Single cell whole cDNA libraries were obtained by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with seminested amplification using 3 ′ constant domain primers followed by 10X Genomics prep. Both single-cell libraries were sequenced in paired-end mode (2 × 150 bp) on an Illumina Hiseq platform. We developed an immunoglobulin alignment tool to enable the analysis of highly mutated IG sequences derived from FL. A consensus sequence was obtained by transcript associated with a unique molecular identifier (UMI) for every cell. Then, the presence of variants in the IG by position in a particular cell was annotated. These observations were filtered using quality parameters (read depth => 25 , frequency of the event => 20%, UMIs supporting every variant => 5).ResultsWe analyzed an average of 926 cells per case. Our data confirms at the single cell level previous reports on high clonal heterogeneity and hypermutation rate (mean 18.7%) in FL. When analyzing intracellular heterogeneity we observed single FL cells expressing simultaneously transcripts derived from the same immunoglobulin VDJ rearrangement but displaying high confidence single nucleotide variants. After applying strict filtering strategies to account for potential technical issues we defined the occurrence of “SHM snapshot events” when a single cell displayed a set of transcripts from a particular VDJ rearrangement with and without the occurring single nucleotide variant. Such events were detected in 8 of the 14 FL analyzed samples. In those 8 samples, 114 events were observed in which two different transcripts of one specific IG were found within a single cell. AID-related motifs (WRCY, WA and RCG) were found in 45% of the SHM snapshot events. SHM snapshot events were undetectable in CLL (5 samples) and 3 SHM snapshot events were detected in MBL (2 samples). In addition, the occurrence of SHM snapshot events was significantly associated with AID expression (Fisher exact test, p-value < 0.001). On the other hand, AID expression was not detectable on CLL and MBL samples.ConclusionHere we report for the first time the occurrence of ongoing somatic hypermutation at a single cell level in FL. The simultaneous detection of both the pre and post mutation IG mRNA transcripts within a single cell may be indicative of SHM occurring more recently than the lifespan of mRNA transcript. The detection of this phenomenon in 57% of FL samples and its association with AID expression suggests that AID-induced mutagenesis may be acting at a much higher rate than expected. This work highlights the role of AID in shaping the tumor heterogeneity in FL and the need to further understand the role of this enzyme in lymphomagenesis and tumor progression. DisclosuresNo relevant conflicts of interest to declare.

Fully Automated Workflows Quantify and Report Key T-Cell and B-Cell Receptor Biomarkers Relevant to Immuno-Oncology and Heme-Oncology Research

BackgroundT-cell and B-cell repertoire analysis is used in oncology research, to understand the etiology of complex disease phenotypes, for the identification of biomarkers predictive of disease burden, outcome, and response to treatment, and for research in diagnosis and recurrence monitoring. Key predictors include secondary and tertiary repertoire features not reported by existing sequencing software solutions. For example, due to ongoing somatic hypermutation in mature B-cell receptors, the underlying sequence of a given clone can accumulate base differences and appear as several distinct clones with smaller frequencies, thereby hampering the ability of analysis software to detect its presence as a single dominant clone with the highest frequency. This has particularly detrimental implications for research in disorders such as follicular lymphoma and may require clonal lineage analysis for proper mitigation. Therefore, to aid the downstream analytics of biomarker identification and the study of complex disease, we developed fully automated analysis solutions that directly compute and report several key features (clonal lineage, amongst several others described below) pertinent to this area of research.ResultsWe developed the Oncomine™ TCR Beta-SR, TCR Gamma-SR, BCR IGH-SR and BCR IGKL-SR workflows on Ion Reporter™ to characterize T-cell (β, γ chains) and B-cell (heavy and light (κ, δ) chains) repertoires. These workflows generate output tables and visualizations for primary repertoire features such as detected clones (viz., unique rearrangements in the receptor DNA sequence), their frequencies, as well as their somatic hypermutation levels in the case of B-cells (Figure 1a & 1b) for clonality assessment and rare clone detection. The software also quantifies and reports several secondary and tertiary repertoire features in a sample, such as clonal diversity, evenness of the clonal population, and B-cell lineage groupings useful in identifying related sub-clones. It includes spectratyping format plots to simultaneously assess the above features as a function of v-gene usage and CDR3 length combinations (Figure 1c & 1d), thereby providing users a complete snapshot of the repertoire, and also the capability to quickly determine CDR3 lengths and V-gene usage of highly expanded or mutated clones. A separate CDR3 lengths histogram is included, as well as a heatmap that depicts the distributions/intensity of Variable-Joining gene combinations (Figure 1e & 1f).Furthermore, the TCR workflows also report (i) convergence frequencies (fraction of clones with different nucleotide sequences, but identical amino acid sequences), and (ii) haplotype grouping for an analyzed sample, based on V-gene allele genotyping and clustering (Figure 1g). In addition, the long read Oncomine™ BCR IGH-LR workflow uniquely reports the isotype class for every detected clone, and includes a visualization of total reads, clones and lineages in the sample represented by isotype (Figure 1h).ConclusionThe Oncomine™ immune repertoire workflows for T-cell and B-cell receptor sequencing were designed to be of high utility in distinct areas of malignancy research, and we expect them to greatly simplify complex downstream analyses. The unique capabilities of the workflows to automatically report secondary and tertiary repertoire features such as (i) clonal lineages for improved dominant clone detection in blood cancers, (ii) TCR clone convergence for prediction of response to immune checkpoint inhibitors [1,2], (iii) TCR haplotype grouping for evaluation of risk factors for autoimmunity and immune-related adverse events [3], and (iv) isotype classification in BCRs for studying pan-cancer immune evasion mechanisms, demonstrate the clear advantages of using these automated workflows over other existing solutions.For research use only.References1) Looney TJ et al. (2020) TCR Convergence in Individuals Treated With Immune Checkpoint Inhibition for Cancer. Front. Immunol. 10:2985.2) Naidus et al. (2021) Early changes in the circulating T cells are associated with clinical outcomes after PD-L1 blockade by durvalumab in advanced NSCLC patients. Cancer Immunology, Immunotherapy 70:2095-21023) Looney TJ et al. (2019) Haplotype Analysis of the T-Cell Receptor Beta (TCRB) Locus by Long-amplicon TCRB Repertoire Sequencing. Journal of Immunotherapy and Precision Oncology. 2 (4): 137-143. [Display omitted] DisclosuresSarda: Thermo Fisher Scientific: Current Employment. Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Looney: Thermo Fisher Scientific: Ended employment in the past 24 months; Singular Genomics: Current Employment. Hyland: Thermo Fisher Scientific: Current Employment.

Higher-order immunoglobulin repertoire restrictions in CLL: the illustrative case of stereotyped subsets 2 and 169

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% of all cases of 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 cases of 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. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the 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 immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21–bearing CLL subset 169 immunoglobulin 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 immunoglobulin features that can be explained only in the context of shared functional selection.

Molecular histogenesis of posttransplantation lymphoproliferative disorders

AbstractPosttransplantation lymphoproliferative disorders (PTLDs) represent a serious complication of solid organ transplantation. This study assessed the molecular histogenesis of 52 B-cell monoclonal PTLDs, including 12 polymorphic PTLDs (P-PTLDs), 36 diffuse large B-cell lymphomas (DLBCLs), and 4 Burkitt/Burkitt-like lymphomas (BL/BLLs). Somatic hypermutation (SHM) of immunoglobulin variable (IgV) genes documented that most monoclonal B-cell PTLDs (75% P-PTLDs, 91.3% DLBCLs, 100% BL/BLLs) derive from germinal center (GC)-experienced B cells. B-cell lymphoma 6 (BCL6) mutations occurred in 25% P-PTLDs, 60.6% DLBCLs, and 75.0% BL/BLLs. A first histogenetic category of PTLDs (31.2% DLBCLs) express the BCL6+/multiple myeloma oncogene-1 protein (MUM1-/+)/CD138- profile and mimic B cells experiencing the GC reaction, as also suggested by ongoing SHM in a fraction of these cases. A second subset of PTLDs (66.7% P-PTLDs and 31.2% DLBCLs) display the BCL6-/MUM1+/CD138- phenotype and mimic B cells that have concluded the GC reaction. A third histogenetic category of PTLDs (25.0% P-PTLDs and 31.2% DLBCLs) shows the BCL6-/MUM1+/CD138+ profile, consistent with preterminally differentiated post-GC B cells. Crippling mutations of IgV heavy chain (IgVH) and/or IgV light chain (IgVL) genes, leading to sterile rearrangements and normally preventing cell survival, occur in 4 DLBCLs and 1 BL/BLL that may have been rescued from apoptosis through expression of Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1). Overall, the histogenetic diversity of monoclonal B-cell PTLDs may help define biologically homogeneous categories of the disease. (Blood. 2003;102: 3775-3785)

Grade I, II and III Follicular Lymphomas Express Ig VH Genes with Different Patterns of Somatic Mutation

Follicular lymphoma (FL) is an indolent, B-cell, non-Hodgkin’s lymphoma with varying cytological appearance and clinical behavior. The genetic hallmark of FL is the t(14;18) translocation, and as a germinal center derived entity it is also characterized by somatic hypermutation of the immunoglobulin heavy chain (IgH) gene. In an attempt to correlate this molecular signature with the cytological grading of FL, we have analyzed the IgH variable (IgVH), regions in all cytological grades of FL. Four FL cases showing t(14;18) translocation were classified into grade I-III categories according to the current WHO guidelines. The IgVH gene segments were PCR-amplified, sequenced, and compared to their respective germline IgVH sequences. The neoplastic cells of grade I and II FLs revealed clonally related, but highly divergent IgVH gene sequences indicating the ongoing nature of somatic hypermutation. Grade III FL also showed extensive presence of somatic hypermutation, but these mutations were not associated with intraclonal divergence. Thus, these results suggest that grade I-II and grade III FL may represent different biological entities. The presence of ongoing somatic hypermutation of IgVH sequences in grade I and II FLs is compatible with direct follicular origin of these tumor cells, contrasting the homogenous, stable clones of grade III FL resembling a post-follicular stage of B-cell development. Our findings demonstrate that contrary to the three tiered cytological grading, molecular features of IgH genes classify FL into two distinct subcategories. These studies also suggest that with progression FL gains post-follicular–like molecular features and becomes independent of the germinal center microenvironment.

Clonal lineage analysis of CLL research samples by IGH chain sequencing to reveal novel CLL subgroups defined by ongoing class-switch recombination and somatic hypermutation.

e20001 Background: B cell somatic hypermutation (SHM) and class switch recombination (CSR) are mechanistically related but distinct processes requiring precisely targeted generation and repair of single and double strand DNA breaks. Within the context of chronic lymphocytic leukemia (CLL), the presence of ongoing SHM or CSR may reveal the functionality of DNA damage repair pathways, with potential relevance to therapeutic strategies involving the generation of DNA breaks or inhibition of DNA repair machinery. Here we apply clonal lineage analysis of IGH chain sequencing data to evaluate CSR and SHM in a cohort of CLL and splenic marginal zone lymphoma (SMZL) research samples. We present evidence of ongoing CSR and SHM in a significant subset of samples. Methods: Multiplex primers targeting the IGH framework 1 region and isotype region (Oncomine IGH-LR assay; detection of all nine isotypes) were used for IGH repertoire sequencing via the Ion Gene Studio S5 from 25ng peripheral blood total RNA derived from 63 individuals with CLL and 4 individuals with SMZL. Clonotyping and clonal lineage analysis was performed by Ion Reporter, whereby clonal lineages are defined as sets of unique rearrangements having a shared variable and joining gene, the same CDR3 length, and a minimum CDR3 nucleotide similarity of 85%. Ongoing CSR was defined as the presence of IgM/IgD and at least one switched isotype (IgG, IgA, or IgE), or a combination of switched isotypes, within the same lineage. Ongoing SHM was defined as the presence of subclones that differ within the VDJ region sequence compared to other clonal lineage members. Results: 11/68 cases showed evidence of ongoing CSR or SHM. Of the 57 cases showing no evidence of ongoing CSR or SHM, variable gene mutation analysis revealed the presence of three distinct subgroups having either no SHM, intermediate SHM (average 98% sequence identity) or high SHM &lt; 94% identity). 3 of 4 SMZL cases showed evidence of ongoing CSR or SHM. Conclusions: These results reveal previously underappreciated heterogeneity within CLL and suggest the subdivision of CLL based on a combination of IGHV mutation level and presence of ongoing SHM or CSR. The described heterogeneity may serve as a valuable criterion for stratifying CLL patients in the future.

IGHV sequencing reveals acquired N-glycosylation sites as a clonal and stable event during follicular lymphoma evolution

AbstractFollicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells' dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

VH CDR3-Focused Somatic Hypermutation in CLL IGHV-IGHD-IGHJ Gene Rearrangements with 100% IGHV Germline Identity

Classification of patients with chronic lymphocytic leukemia (CLL) based on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the sequence of the rearranged IGHV gene excluding the VH CDR3. This is mostly due to the difficulty in discriminating actual SHM from random nucleotides added between the recombined IGHV, IGHD and IGHJ genes. Hence, this approach may underestimate the true impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions i.e. the VH CDR3. Relevant to mention in this respect, studies from our group in CLL with mutated IGHV genes (M-CLL), particularly subset #4, have revealed considerable intra-VH CDR3 diversity attributed to ongoing SHM. Prompted by these findings, here we investigated whether SHM may also be present in cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3), focusing on two well characterized stereotyped subsets i.e. subset #1 (IGHV clan I/IGHD6-19/IGHJ4) and subset #6 (IGHV1-69/IGHD3-16/IGHJ3). These subsets carry germline-encoded amino acid (aa) motifs within the VH CDR3, namely QWL and YDYVWGSY, originating from the IGHD6-19 and IGHD3-16 gene, respectively. However, in both subsets, cases exist with variations in these motifs that could potentially represent SHM. The present study included 12 subset #1 and 5 subset #6 patients with clonotypic IGHV genes lacking any SHM (100% germline identity). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified by subgroup-specific leader primers and a high-fidelity polymerase in order to ensure high data quality. RT-PCR products were subjected to paired-end NGS on the MiSeq platform. Sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis was undertaken using an in-house purpose-built bioinformatics pipeline. Rearrangements with the same IGHV gene and identical VH CDR3 aa sequences were defined as clonotypes. Overall, we obtained 1,570,668 productive reads with V-region identity 99-100%; of these, 1,232,958 (mean: 102,746, range: 20,796-242,519) concerned subset #1 while 337,710 (mean: 67,542, range: 50,403-79,683) concerned subset #6. On average, 64.4% (range: 1.7-77.5%) of subset #1 reads and 49.2% (range: 0.7-70%) of subset #6 reads corresponded to rearrangements with IGHV genes lacking any SHM (100% germline identity). Clonotype computation revealed 1,831 and 1,048 unique clonotypes for subset #1 and #6, respectively. Subset #1 displayed a mean of 157 distinct clonotypes per sample (range: 74-267), with the dominant clonotype having a mean frequency of 96.9% (range: 96-98.2%). Of note, 44 clonotypes were shared between different patients (albeit at varying frequencies), including the dominant clonotype of 11/12 cases, which was present in 2-6 additional subset #1 patients. Subset #6 cases carried a higher number of distinct clonotypes per sample (mean: 219, range: 189-243) while the dominant clonotype had a mean frequency of 95.6% (range: 94.5-96.5%). Shared clonotypes (n=30) were identified also in subset #6 and the dominant clonotype of each subset #6 case was present in 3-5 additional subset #6 patients. Focusing on the VH CDR3, in particular the IGHD-encoded part, the following observations were made: (1) in both subsets, extensive intra-VH CDR3 variation was detected at certain positions within the IGHD gene; (2) in most cases, the observed aa substitutions were conservative i.e. concerned aa sharing similar physicochemical properties. Particularly noteworthy in this respect were the observations in subset #6 that: (i) the valine residue (V) in the D-derived YDYVWGSY motif was very frequently mutated to another aliphatic residue (A, I, L); (ii) in cases were the predominant clonotype carried I (also in the Sanger-derived sequence), several minor clonotypes carried the germline-encoded V, compelling evidence that the observed substitution concerned true SHM. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, very likely attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. While the prognostic/predictive relevance of this observation is beyond the scope of the present work, our findings highlight the possible need to reappraise definitions ('semantics') regarding SHM status in CLL. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.

Immunoglobulin somatic hypermutation has clinical impact in DLBCL and potential implications for immune checkpoint blockade and neoantigen-based immunotherapies

BackgroundDiffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma.MethodsTo determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens’ immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry.ResultsSHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHMhigh) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell–like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHMhigh groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8+ T cells was significantly lower in IGHV SHMhigh than in SHMlow patients with activated B-cell–like DLBCL, whereas PD-1 expression in CD4+ T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHMhigh than in SHMlow patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM.ConclusionsThese results show for the first time that IGV SHMhigh and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.

Primary Cutaneous Follicle Center Lymphomas (PCFCL) Express Heavily Mutated B-Cell Receptors with Acquired N-Glycosylation Motifs and Lack Ongoing Somatic Hypermutation

Introduction & Objectives: Primary Cutaneous Follicle Center Lymphoma (PCFCL) is a very indolent mature B-cell lymphoma that shares germinal center morphology with follicular lymphoma (FL) but lacks the characteristic t(14;18). Unlike FL, immunohistochemistry fails to detect expression of BCL2, CD10, and immunoglobulin in PCFCL. Therefore, we investigated expression of B-cell receptor (BCR) transcripts to gain insight into the immunobiology of PCFCL.Materials & Methods: Full-length heavy and light chain BCR transcripts of 13 histologically confirmed PCFCL were amplified using ARTISAN PCR and sequenced on the PacBio RSII system. BCR from 4 cases were sequenced to a depth of >2000 sequences per BCR transcript; the remaining cases to a median depth of 1663 sequences (range: 626-5301). BCR from 51 cases of FL and from peripheral B cells of 12 healthy donors were used as controls. Whole genome sequencing (WGS) and RNAseq were performed on 5 PCFCL on the Illumina HiSeq platform.Results: No PCFCL case carried a t(14;18). In addition to previously described CD79B mutations, an L265P mutation in MYD88 was identified in one case, and two PCFCL carried amplifications in chromosome 2 involving the proto-oncogene REL.ARTISAN PCR demonstrated expression of potentially functional VDJ and VJ genes with heavily mutated V regions (VDJ: 5.9-24.0%; VJ: 4.7-17.9%) in all PCFCL cases, which could be confirmed by RNAseq-based de novo BCR assembly. One PCFCL case expressed IgM, another IgA, and the remaining ten cases expressed IgG. PCFCL VDJ carried relatively long heavy chain CDR3 regions with a median of 19 amino acids (versus 17 in healthy donor PBMCs).In contrast to FL, only minimal intraclonal sequence variation (comparable to the known error rate of the used sequencing method) was observed in PCFCL VDJ and VJ sequences, indicating absence of ongoing somatic hypermutation (SHM).VDJ and/or VJ of 11 PCFCL (85%) carried at least one acquired N-linked glycosylation motifs, six PCFCL (46%) at least two, and one case four such motifs. 75% of acquired N-linked glycosylation motifs were found in different positions than the N-linked glycosylation motifs found in FL BCR (Figure). In contrast, only 17.5% and <0.5% of mutated BCR with less than 98% IGV homology to germ-line from healthy donors carried at least one or more acquired N-linked glycosylation motifs, respectively.The PCFCL BCR with the most N-linked glycosylation motifs belonged to the only patient with a lymph node relapse and currently active disease. BCR of PCFCL with an at least partly follicular growth pattern appeared to carry more N-linked glycosylation sites than PCFCL with a strictly diffuse growth pattern (average 1.86 (range: 1-4) versus 0.83 (range: 0-2)), although this difference was not significant (p=0.10). Additionally, 6/7 PCFCL with a follicular growth pattern but none of the PCFCL with a diffuse growth pattern were situated on the scalp (p<0.001).Conclusions: Follicular morphology, class switch recombination, and extensive SHM indicate a shared germinal center B cell origin for both PCFCL and FL. Clonal BCR sequences and previously identified copy number alterations prove that PCFCL represents a neoplastic clonal expansion. However, lack of ongoing SHM indicates that the immune follicles of PCFCL are not fully functional germinal centers. Since ongoing SHM is thought to contribute to lymphomagenesis by targeting non-BCR loci, absence of both ongoing SHM and the t(14;18) may explain the relatively benign clinical course of PCFCL compared to FL. As previously described for FL, continuous BCR stimulation through glycosylation-mediated binding of lectins on resident cells of the follicular microenvironment may play a role in the clonal expansion of PCFCL cells and may orchestrate the follicular microarchitecture in PCFCL. However, the positions of the glycosylated amino acids within the BCR vary between FL and PCFCL and warrant functional studies to clarify their relevance in PCFCL pathogenesis. [Display omitted] DisclosuresSchmidt:Gilead: Honoraria, Other: Travel Grants; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants; Celgene: Honoraria.