BTK and PLCG2 remain unmutated in one-third of patients with CLL relapsing on ibrutinib.

Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet need. Though Bruton tyrosine kinase (BTK) and PLCG2 mutations are associated with ibrutinib resistance, their frequency and relevance to progression are not fully understood. In this multicenter retrospective observational study, we analyzed 98 patients with CLL on ibrutinib (49 relapsing after an initial response and 49 still responding after ≥1 year of continuous treatment) using a next-generation sequencing (NGS) panel (1% sensitivity) comprising 13 CLL-relevant genes including BTK and PLCG2. BTK hotspot mutations were validated by droplet digital polymerase chain reaction (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32 of 49 relapsing cases (65%) carried at least 1 hotspot BTK and/or PLCG2 mutation(s); in 6 of 32, BTK mutations were only detected by ddPCR (variant allele frequency [VAF] 0.1% to 1.2%). BTK/PLCG2 mutations were also identified in 6 of 49 responding patients (12%; 5/6 VAF <10%), of whom 2 progressed later. Among the relapsing patients, the BTK-mutated (BTKmut) group was enriched for EGR2 mutations, whereas BTK-wildtype (BTKwt) cases more frequently displayed BIRC3 and NFKBIE mutations. Using an extended capture-based panel, only BRAF and IKZF3 mutations showed a predominance in relapsing cases, who were enriched for del(8p) (n = 11; 3 BTKwt). Finally, no difference in TP53 mutation burden was observed between BTKmut and BTKwt relapsing cases, and ibrutinib treatment did not favor selection of TP53-aberrant clones. In conclusion, we show that BTK/PLCG2 mutations were absent in a substantial fraction (35%) of a real-world cohort failing ibrutinib, and propose additional mechanisms contributing to resistance.

Different prognostic impact of recurrent gene mutations in chronic lymphocytic leukemia depending on IGHV gene somatic hypermutation status: a study by ERIC in HARMONY.

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3-9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

High surface IgM levels associate with shorter response to ibrutinib and BTK bypass in patients with CLL.

Chronic lymphocytic leukemia (CLL) cells have variably low surface IgM (sIgM) levels/signaling capacity, influenced by chronic antigen engagement at tissue sites. Within these low levels, CLL with relatively high sIgM (CLLhigh) progresses more rapidly than CLL with low sIgM (CLLlow). During ibrutinib therapy, surviving CLL cells redistribute into the peripheral blood and can recover sIgM expression. Return of CLL cells to tissue may eventually recur, where cells with high sIgM could promote tumor growth. We analyzed time to new treatment (TTNT) following ibrutinib in 70 patients with CLL (median follow-up of 66 months) and correlated it with pretreatment sIgM levels and signaling characteristics. Pretreatment sIgM levels correlated with signaling capacity, as measured by intracellular Ca2+ mobilization (iCa2+), in vitro (r = 0.70; P < .0001). High sIgM levels/signaling strongly correlated with short TTNT (P < .05), and 36% of patients with CLLhigh vs 8% of patients with CLLlow progressed to require a new treatment. In vitro, capacity of ibrutinib to inhibit sIgM-mediated signaling inversely correlated with pretherapy sIgM levels (r = -0.68; P = .01) or iCa2+ (r = -0.71; P = .009). In patients, sIgM-mediated iCa2+ and ERK phosphorylation levels were reduced by ibrutinib therapy but not abolished. The residual signaling capacity downstream of BTK was associated with high expression of sIgM, whereas it was minimal when sIgM expression was low (P < .05). These results suggested that high sIgM levels facilitated CLL cell resistance to ibrutinib in patients. The CLL cells, surviving in the periphery with high sIgM expression, include a dangerous fraction that is able to migrate to tissue and receive proliferative stimuli, which may require targeting by combined approaches.

Validation of the EuroClonality-NGS DNA capture panel as an integrated genomic tool for lymphoproliferative disorders.

Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.

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.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study.

Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.

Triggering interferon signaling in T cells with avadomide sensitizes CLL to anti-PD-L1/PD-1 immunotherapy.

Cancer treatment has been transformed by checkpoint blockade therapies, with the highest anti-tumor activity of anti-programmed death 1 (PD-1) antibody therapy seen in Hodgkin lymphoma. Disappointingly, response rates have been low in the non-Hodgkin lymphomas, with no activity seen in relapsed/refractory chronic lymphocytic leukemia (CLL) with PD-1 blockade. Thus, identifying more powerful combination therapy is required for these patients. Here, we preclinically demonstrate enhanced anti-CLL activity following combinational therapy with anti-PD-1 or anti-PD-1 ligand (PD-L1) and avadomide, a cereblon E3 ligase modulator (CELMoD). Avadomide induced type I and II interferon (IFN) signaling in patient T cells, triggering a feedforward cascade of reinvigorated T-cell responses. Immune modeling assays demonstrated that avadomide stimulated T-cell activation, chemokine expression, motility and lytic synapses with CLL cells, as well as IFN-inducible feedback inhibition through upregulation of PD-L1. Patient-derived xenograft tumors treated with avadomide were converted to CD8+ T cell-inflamed tumor microenvironments that responded to anti-PD-L1/PD-1-based combination therapy. Notably, clinical analyses showed increased PD-L1 expression on T cells, as well as intratumoral expression of chemokine signaling genes in B-cell malignancy patients receiving avadomide-based therapy. These data illustrate the importance of overcoming a low inflammatory T-cell state to successfully sensitize CLL to checkpoint blockade-based combination therapy.

Immunoglobulin gene analysis in chronic lymphocytic leukemia in the era of next generation sequencing.

Twenty years after landmark publications, there is a consensus that the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene is an important cornerstone for accurate risk stratification and therapeutic decision-making in patients with chronic lymphocytic leukemia (CLL). The IGHV SHM status has traditionally been determined by conventional Sanger sequencing. However, NGS has heralded a new era in medical diagnostics and immunogenetic analysis is following this trend. There is indeed a growing demand for shifting practice and using NGS for IGHV gene SHM assessment, although it is debatable whether it is always justifiable, at least taking into account financial considerations for laboratories with limited resources. Nevertheless, as this analysis impacts on treatment decisions, standardization of both technical aspects, and data interpretation becomes essential. Also, the need for establishing new recommendations and providing dedicated education and training on NGS-based immunogenetics is greater than ever before. Here we address potential and challenges of NGS-based immunogenetics in CLL. We are convinced that this perspective helps the hematological community to better understand the pros and cons of this new technological development for CLL patient management.

Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tailored approaches grounded on immunogenetic features for refined prognostication in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinico-biological differences. Considering this, we assessed prognosis separately within mutated (M-CLL) and unmutated (U-CLL) CLL in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet A M-CLL patients, besides TP53 abnormalities, trisomy 12 and stereotyped subset #2 membership were equivalently associated with the shortest time-to-first-treatment and a treatment probability at five and ten years after diagnosis of 40% and 55%, respectively; the remaining cases exhibited 5-year and 10-year treatment probability of 12% and 25%, respectively. Within Binet A U-CLL patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-treatment (5- and 10-year treatment probability: 78% and 98%, respectively); in the remaining cases, males had a significantly worse prognosis than females. In conclusion, the relative weight of indicators that can accurately risk stratify early-stage CLL patients differs depending on the somatic hypermutation status of the immunoglobulin heavy variable genes of each patient. This finding highlights the fact that compartmentalized approaches based on immunogenetic features are necessary to refine and tailor prognostication in CLL.

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation.

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a 'molecular timeline' since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

Automated shape-based clustering of 3D immunoglobulin protein structures in chronic lymphocytic leukemia.

BACKGROUND: Although the etiology of chronic lymphocytic leukemia (CLL), the most common type of adult leukemia, is still unclear, strong evidence implicates antigen involvement in disease ontogeny and evolution. Primary and 3D structure analysis has been utilised in order to discover indications of antigenic pressure. The latter has been mostly based on the 3D models of the clonotypic B cell receptor immunoglobulin (BcR IG) amino acid sequences. Therefore, their accuracy is directly dependent on the quality of the model construction algorithms and the specific methods used to compare the ensuing models. Thus far, reliable and robust methods that can group the IG 3D models based on their structural characteristics are missing. RESULTS: Here we propose a novel method for clustering a set of proteins based on their 3D structure focusing on 3D structures of BcR IG from a large series of patients with CLL. The method combines techniques from the areas of bioinformatics, 3D object recognition and machine learning. The clustering procedure is based on the extraction of 3D descriptors, encoding various properties of the local and global geometrical structure of the proteins. The descriptors are extracted from aligned pairs of proteins. A combination of individual 3D descriptors is also used as an additional method. The comparison of the automatically generated clusters to manual annotation by experts shows an increased accuracy when using the 3D descriptors compared to plain bioinformatics-based comparison. The accuracy is increased even more when using the combination of 3D descriptors. CONCLUSIONS: The experimental results verify that the use of 3D descriptors commonly used for 3D object recognition can be effectively applied to distinguishing structural differences of proteins. The proposed approach can be applied to provide hints for the existence of structural groups in a large set of unannotated BcR IG protein files in both CLL and, by logical extension, other contexts where it is relevant to characterize BcR IG structural similarity. The method does not present any limitations in application and can be extended to other types of proteins.

Highly similar genomic landscapes in monoclonal B-cell lymphocytosis and ultra-stable chronic lymphocytic leukemia with low frequency of driver mutations.

Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we performed whole-genome sequencing in 11 individuals with monoclonal B- cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.

Drug-perturbation-based stratification of blood cancer.

As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.

Chronic Lymphocytic Leukemia with Mutated IGHV4-34 Receptors: Shared and Distinct Immunogenetic Features and Clinical Outcomes.

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication.Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34-expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes.Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively).Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292-301. ©2017 AACR.