IGHV Research Articles

Immune2vec: Embedding B/T Cell Receptor Sequences in ℝN Using Natural Language Processing

The adaptive branch of the immune system learns pathogenic patterns and remembers them for future encounters. It does so through dynamic and diverse repertoires of T- and B- cell receptors (TCR and BCRs, respectively). These huge immune repertoires in each individual present investigators with the challenge of extracting meaningful biological information from multi-dimensional data. The ability to embed these DNA and amino acid textual sequences in a vector-space is an important step towards developing effective analysis methods. Here we present Immune2vec, an adaptation of a natural language processing (NLP)-based embedding technique for BCR repertoire sequencing data. We validate Immune2vec on amino acid 3-gram sequences, continuing to longer BCR sequences, and finally to entire repertoires. Our work demonstrates Immune2vec to be a reliable low-dimensional representation that preserves relevant information of immune sequencing data, such as n-gram properties and IGHV gene family classification. Applying Immune2vec along with machine learning approaches to patient data exemplifies how distinct clinical conditions can be effectively stratified, indicating that the embedding space can be used for feature extraction and exploratory data analysis.

Repertoire of Rearranged Immunoglobulin Heavy Chain Genes in Russian Patients With B-Cell Lymphoproliferative Diseases

IntroductionImmunoglobulin heavy chain variable region (IGHV) repertoire narrowing could be an evidence for the involvement of a limited set of antigens in the development of lymphomas. For chronic lymphocytic leukemia (CLL) the existence of more than 200 subgroups of tumor IGHV antigen-binding sites, so called “stereotypical” antigen receptors (SAR) has been shown. For others lymphomas the possibility of SARs is also suggested. The aim of this study is to compare the tumor IGHVs and possible SARs in various B-cell malignancies in Russia and other countries. Materials and MethodsThe study included samples of 1800 CLL patients, 52 patients with mantle cell lymphoma, 48 patients with hairy cell lymphoma and 37 patients with splenic marginal cell lymphoma. The nucleotide sequences of the IGHV genes were determined according to ERIC protocol. ResultsIn CLL most common IGHV genes were IGHV1-69, IGHV1-2, IGHV3-30 and IGHV4-34. The most common SARs were CLL#1, CLL#6, CLL#2, CLL#3. In MCL the most common genes were IGHV4-34, IGHV3-21, IGHV3-23. In 5 MCL patients CDR3 sequences were identified matching definitions of a stereotyped. In the half of SMZL patients was identified gene IGHV1-2. Other IGHV genes were much less common. Two pairs of SMZL patients have motives similar to each other. In HCL IGHV repertoire was the most variable, no trends for antigen receptor stereotypy were observed. It was found that SARs are highly disease-specific both at the level of nucleotide and amino acid sequences. ConclusionOur results suggest that antigens crucial for the pathogenesis of B-cell malignancies could be disease-specific. Further studies on extended samples of non-CLL patients concerning the role of SARs in pathogenesis of these diseases may also contribute to the development of new diagnostic and prognostic markers.

Optimal Management of Chronic Lymphocytic Leukemia and Economic Constraints.

In this article, we carry out an overview on the management options available for chronic lymphocytic leukemia (CLL) patients and discuss possible treatment decisions, taking into account the issue of sustainability and availability. Targeted agents have shown to be superior compared with chemoimmunotherapy (CIT) in terms of progression-free survival in high-risk CLL. In the majority of studies, however, continuous treatment was compared with fixed-duration CIT and no overall survival or progression-free survival-2 (time from randomization to second progression or death) advantage could be documented. Meanwhile, a substantial financial burden on both patients and payers has raised issues about affordability and adherence to treatment. Therefore, value-based pricing of new drugs has been used to set up price negotiation policies in several countries, and fixed-duration therapy has shown to be less costly than continuous treatment. Thus, CIT continues to have a role in the treatment of CLL patients with a favorable genetic profile, that is, with a mutated IGHV gene profile and a wild-type TP53. Targeted treatment represents the preferred choice in patients with an unmutated IGHV gene configuration and/or a TP53 disruption, provided that adherence to treatment is guaranteed and bearing in mind that should costly drugs not be available for frontline treatment, new agents can be very effective as first salvage treatment.

A Case of Chronic Lymphocytic Leukemia with Unmutated Variable Region of the Immunoglobulin Heavy Chain Gene with Markedly High Lactate Dehydrogenase Responding to Ibrutinib

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Several prognostic factors exist that help stratify patients into different risk categories. Unmutated status of the immunoglobulin heavy chain (IGHV) gene has been shown to be among the most important high-risk prognostic factors in CLL. Elevated serum level of lactate dehydrogenase has also been shown to be associated with worst outcomes. Recently, the management of CLL has progressed favorably and improved outcomes were seen with the use of the small-molecule Bruton's tyrosine kinase inhibitor, ibrutinib, when compared with traditional chemotherapy. Here, we present a CLL case with an unmutated IGHV gene status presenting with markedly elevated LDH and responding dramatically to ibrutinib.

Abstract 511: Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing

Abstract Background Traditional next generation sequencing methods for quantifying somatic hypermutation (SHM) rely on multiplex primers targeting either the framework 1 (FR1) or Leader regions of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA templates. Ion AmpliSeq primer panels for SHM evaluation were compared using both DNA and RNA input. Performance was compared using SHM values obtained from RNA samples amplified using FR1 variable gene primers in combination with constant gene primers to determine each IGH isotype and subtype in a single PCR reaction. Comparison of SHM frequencies measured from matched RNA and DNA samples were used to determine the feasibility for use of RNA in the study of SHM as well as comparison of the performance of Leader and FR1 gene primers in DNA studies. Methods Two multiplex primer panels were developed for DNA templates. These panels target the Leader or FR1 regions of the IGHV gene and the IGHJ gene. RNA samples were surveyed using FR1 and constant region primers. Comparisons of SHM frequency using 200ng of gDNA and 25ng of RNA from B cell lines and clinical research samples. Sequencing was accomplished using the Ion Gene Studio S5, while clonotyping, isotype determination, and somatic hypermutation analysis was performed using Ion Reporter 5.16 analysis software. Results Both RNA and DNA input assay workflows were able to correctly determine the SHM status of all rearrangements tested. IGHV SHM values were highly concordant between both RNA and DNA approaches. Additionally, SHM values derived from FR1 targeting variable gene primers delivered concordant results compared to Leader targeting variable gene primers when using DNA input across a wide range of SHM frequencies tested. Conclusions These results support the ability of highly multiplexed long-read NGS assays to accurately quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader targeting primers using DNA input. RNA based NGS methods benefit from lower sample requirements as well as the addition of isotype (and subtype) identification, opening new research areas for study of the B cell immune repertoire. Citation Format: Michelle Toro, Geoffrey Lowman, Loni Pickle, Stephanie Ostresh, Mark Andersen, Shrutii Sarda, Chenchen Yang. Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 511.

BRAF and BIRC3 Mutations Stratify a Poor Prognostic Subgroup in FCR Treated Chronic Lymphocytic Leukemia

Abstract Background. The current shift of therapy of chronic lymphocytic leukemia (CLL) towards novel targeted agents mandates the recognition of molecular predictors to identify patients who can still benefit from chemoimmunotherapy and those who should instead be considered for novel targeted agents upfront. In the case of FCR (fludarabine, cyclophosphamide, rituximab), the IGHV mutation status and FISH karyotype stratify low-risk patients carrying mutated IGHV genes and devoid of both del11q and del17p who maximally benefit from such treatment; intermediate-risk patients harboring unmutated IGHV genes and/or del11q in the absence of del17p are a case mix of good and poor responders to FCR; while high-risk patients harboring del17p are unsuitable for chemoimmunotherapy. This model fails to consider the impact of recurrent CLL mutations of potential prognostic relevance, which may be improved by their inclusion. Purpose. We aimed at refining the genetic-based stratification of FCR-treated CLL patients by integrating the mutational profile in a prognostic model together with the IGHV mutation status and FISH karyotype. Methods. A multicenter cohort of 173 (162 with complete molecular data) untreated CLL receiving first-line therapy with FCR in the real-life clinical practice was evaluated by target resequencing. Tumor genomic DNA collected at the time of treatment was analyzed for mutations in the coding exons plus splice sites of CLL cancer driver genes (n=23). Deep next-generation-sequencing (NGS) of the gene panel was performed on the Illumina MiSeq platform (coverage >2000x in >90% of the target). Non-synonymous mutations represented in >10% of tumor allele were called by using VarScan2, and a stringent bioinformatic pipeline was developed to protect against the false call of polymorphisms and sequencing errors. Medical statistics was performed using SPSS version 24.0 and R version 3.3.2. Results. The cohort characteristics and mutational profile were consistent with those reported in CLL receiving FCR as initial treatment. After a median follow-up of 7.2 years, 114 patients progressed, accounting for a median PFS of 4.5 years. Among patients categorized as low-risk by IGHV and FISH status, gene mutations associated with poor prognosis were virtually absent (Fig. 1A). By univariate analysis, none of the cancer driver gene mutations significantly associated with PFS among low-risk patients. Consistently, by recursive partitioning, the proportion of low-risk patients failing FCR early was not explained by the co-occurrence of a cancer driver gene mutation. Among patients categorized as intermediate-risk by IGHV and FISH status, mutations of BIRC3 (HR: 6.452; 95% CI 2.467-16.875; p Download : Download high-res image (256KB) Download : Download full-size image Disclosures Forconi: AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Zaja: Novartis: Honoraria, Research Funding; Janssem: Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria; Takeda: Honoraria; Gilead: Honoraria. Rigolin: Celgene: Honoraria, Other: Advisory Board; Mundipharma: Other: Advisory Board; Gilead: Honoraria. Coscia: Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Tedeschi: AbbVie: Consultancy; Gilead: Consultancy, Other: travel expenses; Janssen: Consultancy, Other: travel expenses. Laurenti: Roche: Other: Advisory Board; Gilead: Other: Advisory Board; Janssen: Other: Advisory Board; Abbvie: Other: Advisory Board. Cuneo: Gilead: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board; Roche: Honoraria, Other: Advisory Board. Foa: AbbVie: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Sandoz: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau. Gaidano: Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Development and Validation of a New Tool for the Characterization of Immunoglobulin Gene Rearrangements in Patients with Chronic Lymphocytic Leukemia By Deep Next Generation Sequencing

Introduction: Chronic Lymphocytic Leukemia (CLL) is a deeply heterogeneous disease from both biological and clinical points of view. This heterogeneity is partly reflected by differences in the mutation status of immunoglobulin variable heavy chain (IgHV) genes. According to this criterion, two main subsets are distinguished by whether CLL cells express an unmutated or mutated, reflecting the stage of normal B cell differentiation from which they originate. These groups also have important clinical differences in terms of clinical outcome. Thus, patients with unmutated IgHV (UM; ≥98% of identity to the germline) genes have a more aggressive disease course and develop more frequently unfavourable genetic deletions or mutations than patients with mutated IgHV (M; <98%).Currently, IgHV Somatic Hypermutation testing is assessed by Sanger sequencing (Sseq), which allows defining the main clone. However, it is not always possible to identify different clones in subclonal patients, which accounts for 20% of cases. Deep next generation sequencing (NGS) of the IgHV locus using consensus primers could broad the availability of deciphering the heterogeneity of CLL.Objective: Validation of a deep next generation sequencing (NGS) tool to analyze the IgHV locus and determinate the presence of multiple productive rearrangements in a series of patients with CLL.Methods: We included 140 samples extracted from peripheral blood of patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2017 (median absolute lymphocytes 11.4x109/L [2,8-239,5x109/L]). Sseq amplification and sequence analysis of IgHV rearrangements were performed on either DNA or cDNA conforming to the updated ERIC recommendations. In any cases we were able to determinate the IGVH identity (68 M and 72 UM) and the stereotypic subset.For IgHV-NGS analysis gDNA was amplified using the Sequencing Multiplex Kit based on based on IGH leader (forward primers) and consensus JH (reverse primer), master mixes and PCR products were purified using magnetic beads, normalized and pooled to create a library for sequencing using a MiSeq equipment. Sequencing data were analyzed using our bioinformatics pipeline.Results:The median of: a) total sequence count analyzed per patient was 2492 (111-17445); b) most abundant unique sequence was 984 (111-7029); c) homology of variable region (IgHV) was 99.3% (87.22-100). We excluded unproductive rearrangement, and found 81.42% (114/140) of patients with unique clone and 18.6% (26/140) with multiple productive rearrangements (Figure 1). The median frequency of the second most abundant productive clone was 9.16% (3.33%-50%) (Table 1).Our tool led to identify of a dominant clonotypic IgHV in all cases (140/140, 100%). When we compared the NGS sequence/mutational status for the most abundant clone with Sseq and for the IgHV status determination, only 1 of 140 (0,7%) showed a major clone with productive rearrangement mutated by Sseq but unmutated by NGS.On the basis of IgVH-NGS subclonal profiles recently described, we identified the following distribution: 5.71% (8/140) of patients presented multiple M clones; 35% (49/140) 1 M clone; 13.6% (19/140) mix of M-UM clones; 37.85% (53/140) 1 UM clone; and 7.85% (11/140) multiple UM clones (Figure 2).Conclusion:The results indicate that our tool is useful to approach IgVH rearrangements since we were able both classified correctly according to IgVH mutational status, and V gene concordant. Moreover it was permitted to provide both the complete V-D-J sequence as well as a relative quantification of different subclones.Acknowledgments: Samples provided by the INCLIVA Biobank (PT13/0010/0004). Supported by: FISS PI 10/02095 and FISS PI 14/02018 [Display omitted] DisclosuresSolano:Neovii: Honoraria.

CLL Stereotyped Subset #4 Igs Acquire Binding to Viable B Lymphocyte Surfaces By Somatic Mutations, Isotype Class Switching, and with the Prerequisite of IG Self-Association

The selection and transformation of a normal B cell into the leukemic state in chronic lymphocytic leukemia (CLL) is mediated at least in part by the structure of the B cell receptor (BCR) immunoglobulin (IG) antigen binding site. A dramatic indication of the role played by BCR is demonstrated by those CLL cases expressing “stereotyped” BCR IGs, characterized by very similar VH CDR3 amino acid sequences often encoded by specific IGHV, IGHD, and IGHJ gene segments. CLL stereotyped subset #4 BCR IGs expressing IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements are prime examples of this. Moreover, these clones are always IgG isotype class switched, and contain IGHV and IGLVκ somatic mutations. Moreover, some of these mutations are shared among patients, and are almost invariably similar physicochemically and located at specific positions in the antigen binding site. In the following study, we evaluated the effects of these unique, acquired structural changes on the (auto)antigenic specificities of subset #4, using 3 mAbs belonging to the subset.Gamma-irradiated Ramos B and Jurkat T cell line cells were incubated with subset #4 IGs under non-permeabilizing conditions, and binding to the B and T cell surfaces was detected by flow cytometry. Unlike most other CLL BCR IGs, the average binding percentage to apoptotic Ramos B cells of subset #4 mAbs was low. Conversely, each subset #4 IG bound live B cells well (CLL 183 - 44.8%; CLL 240 - 55.6%; CLL 342 - 52.0%). Moreover, all 3 IGs exhibited minimum binding to live and apoptotic Jurkat T cells. Next we exposed the subset #4 IGs to viable human B lymphocytes from tonsillectomy samples to confirm that binding was not limited to transformed B cells, and indeed the IGs bound well to CD19+ while minimally to CD3+ tonsil cells. When murine mAbs reactive with appropriate B-cell surface antigens were used to define subpopulations of normal B cells with which the subset #4 IGs interacted, we found that the subset #4 IGs and a non-subset #4 IGHV4-34-expressing CLL case (CLL 141) bound viable, naïve human B cells as expected from other studies. However, it became clear that the subset #4 IGs also interacted with viable memory B cells (CLL 183 - 28.1%; CLL 240 - 34.0%; CLL 342 - 42.7%; CLL 141 - 11.8%), a subpopulation that CLL 141 do not bind; the latter similar to IGHV4-34-expressing IGs in other disease states. Furthermore, although DNA and the “i” and “I” carbohydrate antigens are common targets of IGHV4-34 utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively, the subset #4 Igs did not bind to either of these autoantigens when tested by ELISA or glycan binding arrays, respectively.Strikingly, subset #4 IG binding to viable Ramos B cells and memory B lymphocytes depended on an asparagine (N) to aspartic acid (D) mutation at position 66 of FR3 in the rearranged IGKV2-30 gene (N vs. D: 5.26% vs. 55.6%); this amino acid residue is shared by many other clones in this subset and comes about by somatic hypermutation (SHM). Interestingly and consistent with the previous finding, another focused mutation of this subset, glutamic acid (E) at position 28 of FR1 in the rearranged IGHV4-34 (G28E), was responsible for the loss of DNA binding and for the diminished I/i glycan binding.Finally, binding to viable Ramos B cells required the presence of the IgG isotype characteristic of this subset, since reactivity with Ramos B lymphocytes disappeared when the stereotyped antigen-binding site was linked to IgM (183, 240, 342 IgG vs. IgM: 44.8%, 55.6%, 51.2% vs. 24.5%, 1.85%, 7.02%, respectively). This requirement for IgG isotype binding to live Ramos B cells is likely due to the documented subset #4 self-association which requires the gamma isotype, since the change of a single amino acid at VH FR1 (E16HA), which disrupts self-association, eliminated binding to Ramos B lymphocytes.Together our findings indicate that distinct autoantigens mediate the positive and negative selection of the structural elements acquired by SHM and isotype class switching that are characteristic of this unique subset of stereotyped BCR IGs. Moreover, our data show that the reactivity of subset #4 IGs with viable B-cell surface requires the self-association of these IGs and, therefore, strongly suggests that viable B-cell surface binding is mediated by a non-conventional binding site created by self-associated surface membrane bound IGs. DisclosuresKolitz:Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy; Gilead, Magellan, and Novartis: Honoraria; Celgene, Jazz: Equity Ownership. Stamatopoulos:Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

IGHV-D-J Repertoire Analysis Obtained By High-Throughput Sequencing of CD5+ B-Cells in CLL Patients: Prognostic Markers and Disease Development Implications

Chronic lymphocytic leukemia (CLL) is a monoclonal B-cell disorder that derives from a mature lymphocyte that can differ in IGHV mutation status: IGHV -unmutated (U-CLL) and IGHV -mutated (M-CLL). Because in the vast majority of patients the IGHV mutation status of the clinically-appreciated clonal sequence remains the same over time when defined by Sanger sequencing, it has been assumed that IGHV-D-J intraclonal diversification does not occur. However, a few studies using Sanger sequencing have indicated that the leukemic clone is not monolithic and can display varying levels of intraclonal heterogeneity(Bagnara et al., 2006; Gurrieri et al., 2002; Volkheimer et al., 2007). Since IGHV-D-J intraclonal diversity is a function of the frequency of variants in relation to the main clone, we used a high-throughput deep sequencing technique to robustly assess intraclonal diversification and evolution of the CLL B lymphocyte. This approach also allowed us to identify and analyze the IGH repertoire of the remaining normal B lymphocytes in the blood expressing CD5.From the PBMC of 37 CLL patients, CD5+ B cells were isolated and full IGHV-D-J sequencing performed from mRNA and sequenced with Illumina MiSeq. Unique Molecular Barcodes (UMI) were incorporated before PCR amplification to eliminate the effects of PCR errors, thereby providing more quantitative data, and rigorous error correction. The latter provides high-quality sequences that permit accurate studying of somatic mutations indicative of intraclonal diversification. Raw reads were processed with a custom pipeline built with pRESTO (Vander Heiden et al., 2014); IGHV, IGHD, and IGHJ gene attribution was performed with IMGT/HighV-QUEST; clonal families were identified with Change-O(Gupta et al., 2015); and data were analyzed with R custom scripts. To make description of findings clear, we refer to the IGHV-D-J rearrangement identified at diagnosis by Sanger sequencing as the “CLL clonotype” and the precise nucleotide sequence of this IGHV-D-J rearrangement as “CLL clone”. Moreover IGHV-D-J rearrangements other than the leukemic one are referred to as “non-leukemic clonotypes”.We observed that CLL clonotypes studied ex vivo without any stimulation exhibit substantial intraclonal diversity of the leukemic IGHV-D-J rearrangement, defining ongoing somatic mutations. This was the case for U-CLL and M-CLL clones. Moreover 84% of M-CLL cases evolved from a precursor differing in IGHV mutational status. Another indication of clonal evolution was branching complexity of phylogenetic trees within the evolving clone. In ~40% of the studied cases, the subclonal variants exhibit a simple branching structure, while in the remaining ~60% a number of subclones form a complex branching structure. Although this intraclonal diversity was substantial, in most instances the CLL clone outstripped these considerably. However in ~30% of CLL cases, we identified at least 1 expanded subclonal variant of the CLL clone that represented ≥ 1% of the leukemia-related IGHV-D-J sequences. Notably, these CLL patients experienced the shortest times to first treatment (P <0.01).Among the CD5+ B-cells with non-leukemic clonotypes, we identified a significant expansion of one or more clonotypes. In ~23% of CLL cases, the single most expanded non-CLL clonotype represented ≥1% of the total. Moreover, these could represent up to 40% of the total IGHV-D-J transcripts sequenced. If the CD5-derived, non-leukemic clonotypes represented ≥0.75% of the total transcripts, these patients associated with longer time to first treatment (P <0.02). Moreover Finally, the CD5-derived, non-leukemic clonotypes fell into a known CLL stereotyped IGHV-D-J sequence at a frequency 10 times higher than found in normal PBMCs.In summary, we document that CLL clones diversify in vivo, accumulating IGHV-D-J mutations that generate clonal complexity and subclonal expansion not previously appreciated. This, occurring in both U-CLL and M-CLL, directly correlates with time to first treatment, and may serve as an indicator of DNA aberrations occurring genome-wide (“genetic instability”) likely reflecting the action of the DNA mutator, activation-induced deaminase that can also lead to genomic changes outside V-gene loci. Finally, the common presence of secondary expanded clones, unrelated to the CLL clone, also has prognostic significance and may give insight into the development of CLL. DisclosuresNo relevant conflicts of interest to declare.

Different Time-Dependent Risk for Disease Evolution in Chronic Lymphocytic Leukemia with Distinct IGHV Gene Somatic Hypermutation Status

A great challenge for early stage patients with chronic lymphocytic leukemia (CLL) is to determine if and, consequently, when they will require treatment, highlighting the need for robust prognostic assessment. Today, immunogenetic and genomic features are considered essential for accurate risk stratification in CLL. However, when utilizing such biomarkers, the prognosis is assessed assuming stable predictability over the disease course. Here, we assessed the risk for CLL evolution overtime by evaluating time-to-first-treatment (TTFT) for 2366 CLL patients not requiring treatment at the time of diagnosis (Binet A: n=1900, 80.3%/Binet B: n=287, 12.1%), hypothesizing that risk evolution might depend on both time and the biological characteristics of the clone. Based on the somatic hypermutation status (SHM) of the immunoglobulin heavy variable genes, 1364 (58%) and 1002 (42%) patients were classified as mutated (M-CLL) and unmutated CLL (U-CLL), respectively. Regarding genomic subgroups of CLL, we focused on high risk patients carrying aberrations within the TP53 gene (TP53 abn, deletion of chromosome 17p and/or TP53 mutations) and low risk patients exhibiting isolated deletion of chromosome 13q or normal FISH according to the Döhner hierarchical model (del(13q)/normal FISH).First, we estimated the risk overtime by the hazard curve. This enables to better visualize changes in risk overtime compared to a Kaplan-Meier curve by showing the estimated proportion of cases receiving treatment in a defined interval, given that a person is still treatment-free at the start of this interval. An interpolation method was then used to estimate the values of the hazard curve at each distinct year from the time of diagnosis, and the corresponding differences in hazard values between M-CLL and U-CLL. These differences constituted the basis for the overtime analysis. In particular, the overall time frame was divided in 5-year intervals, and the distributions of differences in hazard were statistically compared between consecutive 5-year intervals. P-values less than 0.05 would indicate different evolution for the hazard curve overtime for the M-CLL and U-CLL.First, we assessed all Binet A patients, regardless of their genomic aberrations background, and found significant differences between M-CLL vs U-CLL with p[0,5]Vs(5,10]=0.006, p(5,10]Vs(10,15]=0.009, p(10,15]Vs(15,20]=0.009, p(15,20]Vs(20,25]=0.014, and p(overall)<0.001, reflecting the constant decrease of the distance between the M-CLL and U-CLL hazard curves (Fig 1a), deemed as statistically significant between consecutive 5-year intervals. Next, we focused on different genomic risk subgroups and found that del(13q)/normal FISH patients followed the M-CLL/U-CLL rule with p[0,5]Vs(5,10]=0.006, p(5,10]Vs(10,15]=0.009, p(10,15]Vs(15,20]=0.009, and p(overall)<0.001, indicating little additional impact on risk evolution beyond that dictated by the SHM status (Fig 1b). In sharp contrast, within TP53 abn patients we found p[0,5]Vs(5,10]=0.006, which reflects the statistically significant increase of the distance between the two hazard curves for M-CLL and U-CLL after the 5th year (Fig 1c), hence indicating decreasing risk for TP53 abn M-CLL and, vice versa, constantly increasing risk for TP53 abn U-CLL after the 5th year. For Binet B patients, we did not observe any significant differences with p[0,5]Vs(5,10]=0.2, highlighting a similar hazard evolution for M-CLL and U-CLL (Fig 1d). Low numbers precluded assessing the impact of genomic aberrations within this subgroup of patients.In conclusion, within all early stage patients as well as in subgroup analysis of high or low genomic risk Binet A patients, M-CLL was found to display a rather stable overtime risk for TTFT, independently of the presence of TP53 abn. This sharply contrasted U-CLL patients, where the overtime risk for TTFT was clearly influenced by the genomic background since del(13q)/normal FISH patients showed a similar pattern with M-CLL whereas TP53 abn patients exhibited a continuously increasing risk for disease progression. From a different standpoint, these findings corroborate the concept that the precise prognostic value of TP53 abn depends on the biological background of the CLL clone that is altogether different in patients with distinct SHM status. [Display omitted] DisclosuresStrefford:Roche: Research Funding. Delgado:Abbvie, Jansen, Gilead, Roche: Consultancy, Speakers Bureau. Stavroyianni:Janssen Pharmaceuticals: Honoraria; Gilead Sciences: Honoraria; Takeda: Honoraria. Gaidano:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Roche: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Novartis SA: Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

BCR Unresponsiveness and 13q14 Duplication Underpin Spontaneous Disease Regression in Chronic Lymphocytic Leukemia

Spontaneous regression of chronic lymphocytic leukemia occurs in less than 2% of cases and the mechanisms that underlie this rare event remain largely unknown. We reviewed 1425 patients with CLL across 4 centers and identified 20 who underwent spontaneous disease regression without treatment over a median period of 8.6 years. This included 12 patients with complete and 8 with partial (> 50%) resolution of lymphocytosis and clinical features. Residual circulating CLL cells remained detectable in all cases and phenotypic, functional and genomic analyses were performed on the residual CLL clone, as well as stored cells where available, to investigate potential mechanisms underlying regression.IGH sequence analysis of sequential samples confirmed that the CLL clone was identical between the time of diagnosis and regression (n=6). Moreover, all cases showed somatic mutation of the IGHV gene and VH3 or VH4 usage was seen in 17 cases. Flow cytometric analysis of spontaneous regression cases (n=17) showed that Ki-67 and CD49d expression was markedly decreased compared to stable indolent disease (n=54) and progressive disease (n=40) whereas CXCR4 and FasR expression was increased. No difference was observed with respect to the expression of CD38, ZAP-70, Bcl-2 and Mcl-1 between spontaneous regression and indolent cases.Sequential analysis revealed that these changes in Ki-67 and CXCR4 expression developed during the period of regression. Furthermore, telomere length analysis demonstrated that tumor cells generally had short telomeres (< 5 kb) after regression and this profile, together with the phenotypic progression, indicates that tumors have undergone an initial period of intense proliferation prior to the development of regression. Importantly, telomerase expression remained high in regression tumors and no evidence of cellular senescence was observed. Analysis of the immune compartment revealed a reduction in T cell and NK cell numbers during spontaneous CLL regression and a modest improvement in T cell function as revealed by increased T cell proliferation and decreased expression of PD-1.The response of tumor cells to stimulation through the B cell receptor (BCR) was also investigated. Surface expression of IgM and IgD was low in regression cases and basal Erk phosphorylation was increased. In addition, these tumors were uniformly unresponsive to stimulation separately with anti-IgM F(ab‘)2 antibodies and anti-IgD F(ab‘)2 antibodies, and combined stimulation with both antibodies. These findings indicate that spontaneously regressed CLL tumors are in a state of profound unresponsiveness to BCR stimulation. Consistent with reduced BCR responsiveness, RNA-seq analysis of sorted CLL cells revealed reduced expression of both MYC and Myc target genes in regression cases as well as downregulation of pathways involved in RNA processing, cellular metabolism and proliferation.Finally, the genomic landscape of tumor cells after regression was analyzed by whole exome sequencing and SNP array analysis on paired germline and CLL samples. CLL genomic complexity was generally low although mutations in TP53, MYD88 or HIST1H1B were observed in 5 tumors. Strikingly, 13q14 duplication was present in 13 of 19 tumors and qPCR analysis showed that this was associated with increased expression of miR-15a/16-1.This is the largest and most comprehensive analysis of spontaneous CLL regression to date and reveals a range of novel features. Tumors that have undergone spontaneous regression express somatically mutated immunoglobulin genes, demonstrate short telomeres with a phenotype of BCR unresponsiveness and have a very high prevalence of 13q14 duplication. This profile suggests a model in which CLL clones undergo substantial proliferation initially which subsequently subside into a state of BCR unresponsiveness and low proliferation. The identification of 13q14 duplication as a recurrent genetic abnormality indicates a potential molecular mechanism for this pattern but it is currently unclear if this lesion is established during tumorigenesis or is acquired during the initial proliferative phase. These findings identify CLL regression as a unique subset of B cell lymphoproliferative disease and suggest that therapies which upregulate the expression of miR-15a/16-1 may represent a mechanism to suppress B-CLL proliferation. [Display omitted] DisclosuresRawstron:Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; BD biosciences: Patents & Royalties; Roche: Consultancy, Honoraria; Gilead: Research Funding. Munir:Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; Gilled: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Hillmen:AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Honoraria, Research Funding; Celgene: Research Funding; GSK: Consultancy, Honoraria, Research Funding.

Genome-Wide DNA Methylation Profiling of Chronic Lymphocytic Leukemia Subsets Carrying Stereotyped B Cell Receptors

In recent years, subsets of chronic lymphocytic leukemia (CLL) patients carrying quasi-identical or stereotyped B cell receptors (BcRs) have been identified that share clinicobiological features and disease outcome. While these stereotyped subsets show distinct gene expression and genomic profiles, the DNA methylation landscape remains largely unexplored. By applying high-resolution 450K methylation arrays, we investigated 176 CLL subset cases belonging to: (i) the clinically aggressive, IGHV-unmutated (U-CLL) subsets #1 (clan I genes/IGKV(D)1-39, n=37) and #8 (IGHV4-39/IGKV1(D)-39, n=21); (ii) the IGHV1-69-expressing U-CLL subsets #3 (n=12), #5 (n=9), #6 (n=22), and #7 (n=12); and, (iii) the indolent, IGHV-mutated (M-CLL) subset #4 (IGHV4-34/IGKV2-30, n=28). In addition, we included subset #2 cases (IGHV3-21/IGLV3-21, mixed mutation status, n=35) that have a poor outcome independent of IGHV mutation status. For comparative purposes, we included a cohort of CLL cases that do not express stereotyped BcRs (‘non-subset’, n=325). These patients were subgrouped according to the recently proposed epigenetic classification of CLL, i.e. poor-prognostic, naïve-like CLL (n-CLL, n=102), favorable-prognostic, memory-like CLL (m-CLL; n=176), broadly corresponding to U-CLL and M-CLL, respectively, and a third intermediate CLL subgroup (i-CLL; n=47), which express borderline mutated IGHV genes and have an intermediate prognosis. Finally, a series of sorted normal subpopulations spanning different stages of B-cell differentiation [precursors (n=22), naïve B cells (n=19) and germinal center/memory B-cells (n=33)] were also included in the analysis. Overall, unsupervised analysis of subset vs. non-subset CLL revealed that all U-CLL subsets clustered with n-CLL, subset #4 clustered with m-CLL, while subset #2 clustered separately with i-CLL (Figure 1). Supervised analysis revealed a limited number of CpG sites that were differentially methylated when comparing each U-CLL or M-CLL subset with non-subset cases. In contrast, almost all subset #2 cases clustered separately from i-CLL in supervised analysis, indicating that this subset might represent a distinct subgroup of i-CLL. We recently demonstrated that the number of epigenetic changes that a tumor acquires, compared to its cellular origin (i.e. ‘epigenetic burden‘), may be a powerful predictor of clinical aggressiveness (Queiros et al, Cancer Cell 2016). When adopting this approach in CLL, comparison of specific subsets vs. their non-subset cases matched by epigenetic subgroup, revealed significant differences in the epigenetic burden amongst the various groupings; for instance, in subset #1 vs. n-CLL (72K vs. 67K, p<0.05) and in subset #2 vs. i-CLL (76K vs. 68K, p=0.001), while no difference was observed between subset #4 vs. m-CLL (83K vs. 82K, p=not significant). Subset #2 cases frequently carry del(11q) and harbor SF3B1 mutations, however, neither the IGHV mutation status nor the presence of del(11q) or SF3B1 mutations had any impact on the epigenetic burden within subset #2. In conclusion, U-CLL and M-CLL subsets generally clustered with n-CLL and m-CLL categories, respectively, implying common cellular origins. In contrast, subset #2 emerged as the first defined member of the i-CLL group, which in turn alludes to a distinct cellular origin and/or pathogenetic process for subset #2 and i-CLL patients. [Display omitted] DisclosuresPapakonstantinou:Janssen Pharmaceuticals: Research Funding; Gilead: Research Funding. Smedby:Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Gaidano:Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Ghia:AbbVie: Consultancy; Adaptive: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy; Roche: Consultancy; Novartis: Research Funding. Stamatopoulos:Novartis SA: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

Reactive Oxygen Species and Metabolic Gene Expression Profiling Further Differentiate CXCR4/CD5 Fractions in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) clones are made up of slow growing, long-lived mature B cells that accumulate in the blood and secondary lymphoid organs. Studies using deuterium exchange into CLL cell DNA of patients drinking heavy water led to the phenotypic classification of intraclonal subfractions that differ in time since last cell division. Cells that have most recently divided, termed the proliferative fraction (PF), are CXCR4DimCD5Bright and older cells that are in a resting state, termed the resting fraction (RF), are CXCR4BrightCD5Dim. These studies suggest a model of the life cycle of a CLL cell that involves exiting of the PF from the tissue into the blood and subsequent homing of the RF from the blood to the tissue to survive and/or recapitulate the division cycle.The CXCL12/CXCR4 axis is central to this trafficking of CLL cells out of and into proliferative niches in the tissue. Moreover, activation of this axis is associated with an increase in the transcriptional activity of sterol response element binding protein-1 (SREBP1) in cancer. SREBP1 regulates genes involved in lipid synthesis and uptake. Since CLL cells preferentially rely on fatty acid (FA) metabolism for energy demands, this concurs with the proposed life cycle of CLL subfractions. CXCL12thereby exerts two functions on the CXCR4BrightCD5DimRF: directional trafficking to the tissue and altering the transcriptome to adapt to the lipid rich environment in the lymph node [LN] that the cells in this fraction will soon encounter. Indeed, our microarray analysis has confirmed that SREBP1a and c are more highly expressed in the RF [1.012x] compared to the PF that express higher levels of the transcriptional targets such as lipoprotein lipase [LPL: 1.201x] fatty acid synthase [FASN: 1.17x] glucose-6-phosphate dehydrogenase [G6PD: 1.505x] and pyruvate kinase [PKM2: 1.433x]. Cancer cells are known to have a concomitance to FA synthesis and oxidation during proliferation. This appears to be true in CLL if we infer that the higher expression of genes involved within the most recently divided cells of the PF are a reflection of how actively dividing cells behave in the LN.Since the main source of reactive oxygen species (ROS) is a by-product of oxidative phosphorylation, we examined the extent that the CLL CXCR4/CD5 subfractions differed in ROS levels as a result of their individual metabolic demands during proliferation. We found that PF has significantly higher levels of ROS compared to the RF, regardless of patient IGHV gene mutation status. Additionally, microarray analysis has revealed that the PF express significantly higher levels of genes involved in response to oxidative stress compared to the RF; these include superoxide dismutase [SOD1: 1.149x], catalase [CAT: 1.462] , hemeoxygenase-1 [HMOX1: 1.695], and glutathione peroxidase [GPX1:1.898] . This suggests that this population is uniquely suited to survive under harsh conditions that result from their distinctive demand for fatty acid oxidation to fuel growth in contrast to normal B-cells. This makes targeting these protective enzymes an attractive therapeutic strategy, as an accumulation of damaging levels of ROS will induce cell death.Finally, we have noted a rare population of cells that are CD5BrightCXCR4Negative that contain the highest levels of ROS in CLL patient PBMCs. Initial studies suggest that these CD5BrightCXCR4Negative cells are more enriched in patient LNs and might represent the cells actively dividing in proliferation centers. If so, those few in the peripheral circulation bearing this phenotype may be the closest link to the tissue resident dividing cells. Consistent with this, concurrent quantitation of ROS and cell cycle analysis by flow cytometry confirmed that higher levels of ROS are present in MEC1cells in S, M, and G2 phases. Current studies involve comparing gene expression profiles of various CXCR4/CD5 fractions, including the CD5Bright CXCR4Negative subset, from the blood and LN based on high ROS and reciprocal densities of CXCR4 and CD5. By sorting this rare population from PB, we may conveniently study those actively dividing CLL cells in the LN in lieu of obtaining rare biopsy samples. DisclosuresBarrientos:AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Janssen: Consultancy. Kolitz:Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy; Celgene, Jazz: Equity Ownership; Gilead, Magellan, and Novartis: Honoraria; Gilead, Novartis, and Seattle Genetics: Other: Travel Support.

Over-Expression of Activation-Induced Cytidine Deaminase in TCL1 Mice Leads to the Development of IGHV-Mutated CLL Clones That Associate with More Aggressive Disease

Introduction. Activation-induced cytidine deaminase (AID) mediates somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes (IG s), both of which are necessary for an effective immune response in normal B cells. An unwanted consequence of AID's role is its contribution to cellular transformation and tumor progression by aberrantly mutagenizing genes outside the IG locus (off-target mutations). In chronic lymphocytic leukemia (CLL), higher levels of IGHV clonal mutations in IGHV-mutated CLL (M-CLL) correlate strongly with better clinical outcomes, while, IGHV -unmutated (U-CLL) cases are associated with worse patient outcome. Paradoxically, U-CLL cells express significantly higher levels of AID than M- CLL cells.Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However, because SHM and CSR occur rarely in the leukemic clones of these animals, TCL1 Tg mice mimic only U-CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address this issue, we developed a TCL1 strain by interbreeding mice over-expressing AID in B lymphocytes (Eμ-TCL1xVκ-AID).Methods. B-cell clonal expansions were identified in spleen cells from 14 TCL1 and 23 TCL1xAID (DT) mice by amplifying cDNAs by PCR using consensus IGHV FR and isotype-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. Ig sequences were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Six DT and 2 TCL1 tumors were transferred to SCID mice (5 mice/donor)Results.Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 21 of 23 DT mice; each animal bore an IgMκ+ clone and 5 also exhibited a concomitant IgGκ+ clone. Approximately 50% used VH1-55, VH11-2, or VH12-3.SHM: The IgM+ TCL1 clones had a mutation frequency of 0.05% for IGHV and 0.09% for IGKV . In contrast, the IGHV and IGKV mutation frequencies were considerably higher in DT mice: IGHV 0.47% in IgM+ and 3.0% in IgG+ clones, and IGKV 0.9% for IgM+ and IgG+ combined. These mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1.However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%) than the TCL1 level (0.05%), it was considerably less than that found in the DT clones using other IGHV s (0.80%). In addition, no mutations were detected in VH1-55 clones from DT mice.IGHV gene use and SHM in clones that underwent CSR: Notably, in 2 of 5 instances the same IGHV-D-J rearrangement was found in IgMκ+ and IgGκ+ clones from the same mice. For the remaining 3, only the IgG+ version was detected. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%).Stereotyped IGHV-D-J and IGkV-Jk rearrangements: Among 32 IgM clones from DT mice, 4 expressed VH11-2 and Vκ14-126 and 8 expressed VH12-3 and Vκ4-91; these rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones.DT mice develop more aggressive disease: we observed shorter median survival in DT mice comparing to TCL1 mice (279 days vs 356 days, P =0.0034)Transfer tumor development: Spleen cells from 6 CLL-bearing mice were transferred into SCID recipients, leading to a selected outgrowth of VH12-3/Vκ4-91 and VH11-2/Vκ14-126 clones. Survival in these mice inversely correlated with IGHV mutations (Figure).Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes: certain IGHV s and IGK Vs are less susceptible to having major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69+ clones in U-CLL patients). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, transfer of DT clones to SCID mice led to more IGHV and IGKV mutations and shorter survival opposite to that in M-CLL patients. Possibly this adverse effect might be a consequence of AID's aberrant action outside the IG loci which is facilitated in DT mice. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

External Stimuli Modulate EZH2 Expression in Chronic Lymphocytic Leukemia. Rationale and Evidence for Synergistic Anti-Tumor Effects of B Cell Signaling and EZH2 Inhibitors

The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). We have previously reported that EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Moreover, we demonstrated that EZH2 pharmacological inhibition induced down-regulation of H3K27me3 levels leading to increased cell apoptosis. Here we sought to investigate if EZH2 expression could be affected by microenvironmental triggering, a key driver of CLL development and evolution.The study group included 10 CLL cases (9 with unmutated and 1 with mutated IGHV genes) that had previously been screened and found responsive to cross-linking of the B cell receptor (BcR) with anti-IgM. CD19+ cells were negatively selected from peripheral blood and stimulated or not through the BcR for 14 hours. Using RQ-PCR we found that stimulation with anti-IgM showed opposing effects on EZH2 mRNA levels, since 5 cases upregulated while the remaining 5 downregulated EZH2. In contrast, stimulation with CpG or CpG/CD40L for 14 hours caused pronounced and consistent upregulation of EZH2 mRNA levels compared to unstimulated cells (FD=8.7, p=0.03 and FD=11.3, p=0.03, respectively; n=6). This effect was also obvious at the protein levels in cells treated with CpG, CD40L and CpG/CD40L for 24 hours, where EZH2 protein was significantly upregulated as revealed by Western blot analysis (FD=3.9, p=0.002; FD=1.7, p=0.005; and FD=2.9, p=0.01, respectively; n=6). In CpG-stimulated cultures, the induction of EZH2 was accompanied by a significant increase in cell viability (FD=2, p=0.002; n=6), supporting the conclusion that EZH2 expression likely contributes to the anti-apoptosis phenotype induced in CLL cells by microenvironmental signals. Prompted by these observations, we then investigated whether B cell signaling inhibition might also modulate EZH2 expression and H3K27me3 levels in vivo by longitudinal profiling of another group of 9 CLL patients that were under ibrutinib treatment. In patients at +1 month under ibrutinib, when, as shown by us and others, CLL cells become anergized through the BcR, we observed a significant decrease in EZH2 protein (FD=3, p=0.0007; n=9) as well a trend for reduced H3K27me3 levels (FD=1.4, p=0.07; n=6) compared to the pre-treatment samples, further highlighting the links between BcR signaling and EZH2 expression and functionality. Next, we assessed ex vivo the effects of ibrutinib on CLL cell viability in cases with differential EZH2 expression [EZH2high (n=7) vs EZH2low (n=6)] and found decreased viability in treated cells compared to control cells (DMSO-exposed) in EZH2high cases (FD=1.9, p=0.0025), with no difference in EZH2low cases.Finally, we examined potential synergistic effects of B cell signaling inhibitors [ibrutinib (IB), idelalisib (IDE)] and EZH2 inhibitors (GSK343, GSK126) in primary CLL cells. To this end, CD19+ cells from 6 CLL cases were first pre-stimulated with CpG for 24 hours and then exposed to either single inhibitors (IB, IDE, GSK343, GSK126) or combinations of B cell signaling and EZH2 inhibitors (IB/GSK343, IDE/GSK343, IB/GSK126). Flow cytometry analysis after 3 days in culture revealed significantly lower CLL cell viability and H3K27me3 levels in CLL cells exposed to combined versus single treatment (FD≥ 1.5, p<0.05).In conclusion, we show that EZH2 expression is affected by signals emanating from CLL microenvironment. Moreover, we provide for the first time evidence that the combination of BcR signaling inhibitors widely used in CLL therapy and EZH2 inhibitors currently tested in phase I/II clinical trials in lymphoid malignancies has synergistic effects, suggesting that targeting both processes might represent a rational and promising therapeutic strategy. DisclosuresStavroyianni:Gilead Sciences: Honoraria; Takeda: Honoraria; Janssen Pharmaceuticals: Honoraria. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Janssen Pharmaceuticals: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Papakonstantinou:Gilead: Research Funding; Janssen Pharmaceuticals: Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Novartis SA: Research Funding.

Autoimmune Cytopenias in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib in Routine Clinical Practice at an Academic Medical Center

INTRODUCTIONThe effects of ibrutinib treatment on the natural history of autoimmune cytopenias (AIC) among chronic lymphocytic leukemia (CLL) patients (pts) treated on clinical trials suggests: i) a low rate of treatment-emergent (TE) AIC (~2%, Rogers, Leukemia 2016; Montillo, BCJ 2017) and ii) improved control of pre-existing AIC at the time ibrutinib is initiated with the ability to discontinue immunosuppressive therapies (~69%, Vitale, Haematologica 2016). Confirmation of these findings in CLL pts treated in routine clinical practice requires further investigation. METHODSBetween 4/2012 and 1/2017, 286 CLL pts treated with ibrutinib outside the context of a clinical trial were seen in the Division of Hematology at Mayo Clinic. The CLL Database and medical records of these pts were reviewed for details of past history of AIC and TE AIC; the diagnosis of AIC was determined by the treating physician. Cox proportional hazards regression models were used to estimate hazard ratios and 95% confidence intervals for the association of history of TE AIC with progression-free (PFS) and overall (OS) survival. Time-dependent Cox regression models were used to look at the association of TE AIC with PFS and OS. Fisher's Exact test was used to test for the association of clinical variables with TE. RESULTSAmong the 286 pts, 242 (85%) pts were treated for relapsed CLL and 44 (15%) pts for previously untreated CLL. The median age at ibrutinib start was 68 years, and 72% were males. Twenty-two percent had TP53 disruption (either del17p or TP53 mutation) and 78% had unmutated IGHV genes. We identified 37/286 (13%) pts with a history of AIC prior to ibrutinib start, including 15 pts with autoimmune hemolytic anemia (AIHA), 11 pts with immune thrombocytopenia (ITP), 5 pts with Evans syndrome, 3 pts with pure red cell aplasia (PRCA), 2 pts with autoimmune neutropenia, and 1 pt with aplastic anemia.Eleven (4%) pts developed TE AIC (including two pts with past history of AIC who developed a different AIC after ibrutinib therapy) including: AIHA (n=4), ITP (n=4), PRCA (n=1), aplastic anemia (n=1), and autoimmune neutropenia (n=1). This occurred after a median of 30 days following the initiation of ibrutinib (range, 8-325 days). Eight pts were able to continue ibrutinib after either temporarily holding ibrutinib (n=6), ibrutinib dose reduction (n=1), or no modification to ibrutinib therapy (n=1). Three of the 11 pts (ITP n=2, aplastic anemia n=1) permanently discontinued ibrutinib as a result of the AIC.Twenty-two of the 37 pts with a prior history of AIC had a remote history of AIC and were not on any AIC treatment and 15/37 pts were receiving AIC directed therapy at ibrutinib start. Among the 15 pts who were on AIC directed therapy at the time of ibrutinib initiation, 6 pts had uncontrolled disease, for which they were receiving ≥20 mg prednisone daily, and 9 pts had controlled AIC on therapy consisting of <20 mg of prednisone daily or equivalent. Five (33%) of the 15 pts on therapy were able to discontinue AIC therapy at a median of 2 months (range, 0-25), an additional 5 (33%) pts were able to reduce the dose and schedule of AIC therapy, and 3 (20%) pts continued AIC therapy with no changes. Two (14%) pts not previously on rituximab were started on the therapy due to continued steroid dependence and inability to taper off steroids. None of these 15 pts had worsening of their underlying AIC as a consequence of initiating ibrutinib therapy. See Figure 1 for temporal relationship of ibrutinib treatment.The median OS and PFS from time of ibrutinib start was not significantly different between pts with a history of AIC and those with no history of AIC (Figures 2 and 3). There was no statistically significant association between age, gender, TP53 disruption, IGHV mutation status, and history of AIC with development of TE or recurrent AIC (p-values all > 0.05). The PFS and OS were not significantly different among pts with TE AIC compared to those pts who did not develop TE AIC. CONCLUSIONIn this large retrospective cohort of ibrutinib treated CLL pts outside the context of clinical trials, we found that 1 out of every 25 pts developed TE AIC. Nonetheless, ~75% pts were still able to continue ibrutinib treatment. Improved control of AIC with discontinuation or de-escalation of AIC treatments in ~67% of pts on AIC directed therapy at ibrutinib start supports previous findings. We did not identify any risk factors for the development of TE AIC in our study. [Display omitted] DisclosuresDing:Merck: Research Funding. Kay:Gilead: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Tolero Corporation: Research Funding. Shanafelt:Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celgene: Research Funding; AbbVie: Research Funding; Hospira: Research Funding; Jannsen: Research Funding; Pharmacyclics: Research Funding. Parikh:AstraZeneca: Honoraria; Pharmacyclics: Honoraria; Pharmacyclics: Research Funding.

CLL-Specific B Cell Receptor Immunoglobulin Stereotypes Are Very Infrequent in Low Count Monoclonal B Cell Lymphocytosis: Implications for Disease Ontogeny

In chronic lymphocytic leukemia (CLL), the existence of subsets of patients with stereotyped B cell receptor immunoglobulin (BcR IG), collectively accounting for ~30% of all CLL, strongly implicates antigen selection in disease ontogeny. That said, it is still inconclusive if and how frequently “CLL-specific” BcR IG gene rearrangements occur in the repertoire of unaffected individuals. This is especially relevant when also considering monoclonal B-cell lymphocytosis (MBL), detected in 3-12% of the general population and characterized by the presence of CLL-like clonal B cells in lower numbers compared to CLL with no evidence of disease. MBL is distinguished into high and low count (HC-MBL and LC-MBL, respectively) with the former considered as a pre-leukemic condition, while the latter having an unclear, if any, link to CLL. Our previous immunogenetic studies have revealed similarities between HC-MBL and CLL, thus setting them apart from LC-MBL that displayed distinct IG gene usage and low incidence of stereotypy. However, these studies were Sanger-based, thus inherently limited regarding sensitivity and accurate quantification.Here we sought to overcome these limitations and obtain a comprehensive view into the composition of the IG repertoire in LC-MBL by NGS immunoprofiling, searching particularly for major CLL IG stereotypes. The study group comprised 23 individuals with CLL-like (CD5+) LC-MBL identified by a standardized flow cytometry protocol. PBMCs were the starting material in 11 cases, whereas sorted MBL cells were studied in the remaining 12 cases; in 9 cases, the normal B cell compartment was also flow sorted. Included in the analysis were also 6 individuals without LC-MBL in whom we sorted different B cell subpopulations, including naïve B cells (n=6) and memory B cells (n=5). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified with leader primers and sequencing libraries were prepared with the TruSeq DNA LT Kit and run on the MiSeq Sequencer using the MiSeq Reagent Kit v3. NGS reads were subjected to an in-house bioinformatics pipeline for clonotype characterization and the identification of CLL-specific BcR IG stereotypes.Overall, 16,857,788 productive filtered-in sequence reads were obtained corresponding to an average of 366,474 reads/sample (range: 67,175-662,994). We identified very few reads (2,470/16,857,788; 0.15%) corresponding to 17/19 major CLL BcR IG stereotypes. Their incidence in LC-MBL did not follow the CLL trend: for instance, the stereotypic IGHV3-21 gene rearrangement defining subset #2, the most frequent in CLL, accounted for only 27/2,470 reads (1.1%) corresponding to major subset stereotypes. Overall, stereotypes were identified in 26/47 samples (55%) from 13/23 LC-MBL and 6/6 non LC-MBL cases. In LC-MBL cases bearing stereotypes the breakdown according to sample type was as follows: (i) PBMCs, n=3 (ii) normal B cell compartment, n=7; (iii) sorted MBL cells, n=5: the stereotypes detected in this particular sample type were present at very low frequencies and concerned rearrangements with unmutated IGHV genes. In individuals without LC-MBL, stereotypes were detected in all 6 samples from naïve B cells versus only 2/5 samples from memory B cells. In general, we did not identify significant associations between specific B cell subpopulations and particular stereotypes, except for the IGHV4-34 rearrangement defining CLL subset #4, an indolent CLL variant with features of BcR-anergized clones, which was found only within naïve B cells.In conclusion, we demonstrate that LC-MBL is immunogenetically distinct from CLL. Major CLL BCR IG stereotypes are very scarce in individuals with or without LC-MBL. This could be taken to imply either that these stereotypes are indeed scarce in the blood of individuals unaffected by CLL or, alternatively, that they might reside within other B cell compartments not assessed in this study. DisclosuresAgathangelidis:Gilead Sciences: Research Funding. Stamatopoulos:Janssen Pharmaceuticals: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Novartis SA: Research Funding. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Research Funding.

Pathogenetic Implications from the Analysis of Immunoglobulin Genes in Chronic Lymphocytic Leukemia of Mediterranean Ancestry: The Lesson of the IGHV3-21 Gene.

Studies of the characteristics and the mutational status of the immunoglobulin (Ig) genes expressed by Chronic Lymphocytic Leukemia (CLL) suggest a potential role for antigenic stimulation in the pathogenesis and/or evolution of the disease. We analyzed IGHV repertoire and mutational status in 553 CLL patients from the Mediterranean area. The most commonly represented IGHV genes mirror the usage of normal B cells, with the exception of IGHV1-18, IGHV3-30.3 and IGHV4-59 that are underrepresented. In addition, the analysis of the IGHV3-21 gene, frequently present in Northern European CLL, showed the following peculiarities: 1) this gene was expressed only in a limited number of cases in our Mediterranean cohort (16/553; 2.9%); 2) based on HCDR3 cluster analysis, IGHV3-21-utilizing cases could be grouped in two different subsets of similar frequency. The first subset (common-HCDR3 subset) included seven cases (out of 16), which carried a very short and virtually identical HCDR3 aminoacid sequence bearing strong similarities to the Northern European IGHV3-21 CLL (Tobin et al, Blood. 2002 Mar 15;99(6):2262–4). All cases used the IGHJ6 gene; 4/7 were unmutated; 6/7 carried the Vλ2-14 (IGLV3-21) light chain gene with a similar LCDR3; all expressed CD38 and had a progressive disease. The second subset (non-homogeneous-HCDR3 subset) included 9 cases and was characterized by long and heterogeneous HCDR3 rearrangements, with diverse IGHJ and IGV light chain gene usage and variable CD38 expression; five out of 9 cases were unmutated; the clinical course of these patients was variable (only 4/9 progressed). The first subset, with the evidence of a similar CDR3 in so many different CLL cohorts, suggests the existence of a specific, yet undefined, antigenic element that may subsequently lead to a more aggressive clinical course. The dramatically different frequency of this subset as compared to Northern Europe suggests that the potential selecting agent may be less frequently encountered in the Mediterranean area. In contrast, the second subset included cases with heterogeneous HCDR3 rearrangements and a variable clinical outcome. This second subset may mirror the heterogeneity and low frequency of expression of IGHV3-21 rearrangements in the normal repertoire of peripheral blood B cells, as shaped by the germline composition, molecular mechanisms and distinct selective influences.

Targeting Bruton’s Tyrosine Kinase in CLL

Targeting the B-cell receptor signaling pathway through BTK inhibition proved to be effective for the treatment of chronic lymphocytic leukemia (CLL) and other B-cell lymphomas. Covalent BTK inhibitors (BTKis) led to an unprecedented improvement in outcome in CLL, in particular for high-risk subgroups with TP53 aberration and unmutated immunoglobulin heavy-chain variable-region gene (IGHV). Ibrutinib and acalabrutinib are approved by the US Food and Drug Administration for the treatment of CLL and other B-cell lymphomas, and zanubrutinib, for patients with mantle cell lymphoma. Distinct target selectivity of individual BTKis confer differences in target-mediated as well as off-target adverse effects. Disease progression on covalent BTKis, driven by histologic transformation or selective expansion of BTK and PLCG2 mutated CLL clones, remains a major challenge in the field. Fixed duration combination regimens and reversible BTKis with non-covalent binding chemistry hold promise for the prevention and treatment of BTKi-resistant disease.

Isolation and characterization of cross-neutralizing coronavirus antibodies from COVID-19+ subjects.

SummarySARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.