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Recurrent amino acid replacements. Counts and distribution per IGHV gene and V-subregion. Respective physicochemical characteristics of the substitutions

IGH rearrangements'intraclonal diversification characteristics. Counts, distribution, hotspot targeting of confirmed/unconfirmed mutations and respective amino acid changes across gene groups and IGHV subregions.

Vardi et al. TCRs in CLL.

Distribution of IGHV4-34 CLL according to IGHV mutational status and subset membership.

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.

Purpose: Prompted by the extensive biases in the immunoglobulin (IG) gene repertoire of splenic marginal-zone lymphoma (SMZL), supporting antigen selection in SMZL ontogeny, we sought to investigate whether antigen involvement is also relevant post-transformation.Experimental Design: We conducted a large-scale subcloning study of the IG rearrangements of 40 SMZL cases aimed at assessing intraclonal diversification (ID) due to ongoing somatic hypermutation (SHM).Results: ID was identified in 17 of 21 (81%) rearrangements using the immunoglobulin heavy variable (IGHV)1-2*04 gene versus 8 of 19 (40%) rearrangements utilizing other IGHV genes (P = 0.001). ID was also evident in most analyzed IG light chain gene rearrangements, albeit was more limited compared with IG heavy chains. Identical sequence changes were shared by subclones from different patients utilizing the IGHV1-2*04 gene, confirming restricted ongoing SHM profiles. Non-IGHV1-2*04 cases displayed both a lower number of ongoing SHMs and a lack of shared mutations (per group of cases utilizing the same IGHV gene).Conclusions: These findings support ongoing antigen involvement in a sizable portion of SMZL and further argue that IGHV1-2*04 SMZL may represent a distinct molecular subtype of the disease. Clin Cancer Res; 22(8); 2032–40. ©2015 AACR.

Univariable and multivariable analysis for TTFT within M-CLL.

Analyzed IGH, IGK/L rearrangements. Annotation characteristics

Network of the aligned IGVH1-2*04 CDR3 sub-regions, depicting the emerging patterns

IGK/L rearrangements'intraclonal diversification characteristics. Counts, distribution, hotspot targeting of confirmed/unconfirmed mutations and respective amino acid changes across gene groups and IGLV subregions.

Purpose: The role of antigen(s) in shaping the T-cell repertoire in chronic lymphocytic leukemia, although relevant for understanding malignant cell interactions with cognate T cells, is largely unexplored.Experimental Design: Here we profiled the T-cell receptor β chain gene repertoire in 58 chronic lymphocytic leukemia patients, focusing on cases assigned to well-characterized subsets with stereotyped clonotypic B-cell receptor immunoglobulins, therefore those cases most evidently selected by antigen (subsets #1, #2, and #4).Results: Remarkable repertoire skewing and oligoclonality were observed, and differences between subsets were noted regarding both T-cell receptor β chain gene usage and the extent of clonality, with subset #2 being the least oligoclonal. Longitudinal analysis of subset #4 cases revealed that although the repertoire may fluctuate over time, certain clonotypes persist, thus alluding to persistent antigenic stimulation. Shared (“stereotyped”) clonotypes were found between different patients, reflecting selection by common antigenic elements. Cross-comparison of our dataset with public databases showed that some T-cell clonotypes may have expanded secondary to common viral infections; however, the majority of clonotypes proved to be disease-specific.Conclusions: Overall, the T-cell receptor β chain repertoire in chronic lymphocytic leukemia is likely shaped by antigen selection and the implicated antigenic elements may concern epitopes that also select the malignant B-cell progenitors or, more intriguingly, chronic lymphocytic leukemia–derived epitopes. Clin Cancer Res; 22(1); 167–74. ©2015 AACR.

Patients with chronic lymphocytic leukemia (CLL) progressing on ibrutinib constitute an unmet clinical need. Though 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 treated with 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 PCR (ddPCR) (0.1% sensitivity). By integrating NGS and ddPCR results, 32/49 relapsing cases (65%) carried at least 1 hotspot BTK mutation and/or PLCG2 mutation(s); in 6/32, BTK mutations were only detected by ddPCR [variant allele frequency (VAF) 0.1-1.2%]. BTK/PLCG2 mutations were also identified in 6/49 responding patients (12%; 5/6 VAF

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. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified.

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.

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.

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.

Mantle cell lymphoma (MCL), a malignancy of B-lymphocytes, has a poor prognosis. It is thus necessary to improve the understanding of the pathobiology of MCL and identify factors contributing to its aggressiveness. Our studies, based on Affymetrix data from 17 MCL biopsies, real-time quantitative polymerase chain reaction data from 18 sorted primary MCL cells and 108 MCL biopsies compared to non-malignant tissue, reveals that GNAZ expression predicts poor clinical outcome of MCL patients (Cox regression, P = 0·014) and lymphocytosis (Mann-Whitney, P = 0·011). We show that GNAZ translates to Gαz protein - a signalling molecule within the G-protein coupled receptor network. Our findings suggest that GNAZ/Gαz contribute to the MCL pathobiology.

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 (c) 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which depletes the intracellular dNTP substrates and thus protects the host (human) cells from replication of viruses such as HIV. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS). In lymphoid malignancies, SAMHD1 gene mutations have been detected in a subset of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) resulting in decreased SAMHD1 mRNA levels and also mantle cell lymphoma (MCL) among B-cell neoplasms as well as in a subset (20%) of T-prolymphocytic leukemia (T-PLL). Therefore, SAMHD1 may play a role in oncogenesis as a tumor suppressor. In addition, SAMHD1 may confer resistance to cytarabine by hydrolysing their active triphosphate metabolites and its high protein levels correlate with poorer clinical outcome in acute myeloid leukemia. The mutation status of SAMHD1 gene and its expression patterns in peripheral T-cell lymphoma types is not known yet. The purpose of this study was to investigate SAMHD1 gene alterations using next generation sequencing and SAMHD1 protein expression in common types of PTCL. Methods: The study group included 81 adult patients with peripheral T-cell lymphomas (PTCL) including 26 patients with ALK+ anaplastic large cell lymphoma (ALCL), 20 ALK- ALCL, 13 angioimmunoblastic T-cell lymphomas (AILT) and 22 PTCL, not otherwise specified (NOS) with pre-treatment, formalin-fixed, paraffin-embedded (FFPE) tumor tissues available for immunohistochemical analysis. Double immunostaining (SAMHD1/CD68) was used to distinguish CD68+ histiocytes from the neoplastic T-cells. The Ventana autostainer and a previously validated monoclonal antibody for SAMHD1 (#A303-691A; Bethyl Laboratories, San Antonio, TX, USA) was utilized. The percentage of SAMHD1-positive cells was calculated by counting at least 500 tumor cells in each case. In a subset of 28 PTCLs, next generation sequencing (NGS) was performed using FFPE tissues and an enriched custom TruSight gene panel of 52 genes relevant to lymphoma biology. In addition, 3 control tissue samples were included in the analysis. The analysis pipeline was based on GATK best practices guidelines and all variants were annotated using Ensembl VEP v94.5. Freedom from progression (FFP) and overall survival (OS), were the clinical endpoints. Survival analyses were performed using the Kaplan-Meier method (log-rank test). Results: The expression level of SAMHD1 (percentage of positive neoplastic T-cells) varied significantly with AILT showing the highest level (median percentage 80%) as compared to ALK+ ALCL that showed the lowest level (median percentage 40%) of SAMHD1 expression (p=0.019, Kruskall-Wallis test). SAMHD1 mutations were detected for the first time in a subset of PTCL including 4/11 (36%) ALK+ ALCL, 1/5 (20%) ALK- ALCL, 3/6 (50%) AILT and 2/5 (40%) PTCL, NOS. The SAMHD1 gene alterations included missense mutations, nonsense (stopcodon) and splice region mutations. Importantly, reduced level (low percentage of positive tumor cells) of SAMHD1 protein expression was significantly associated with the presence of SAMHD1 mutations. More specifically, the median percentage of SAMHD1+ neoplastic T-cells was 80% in the PTCL group with wild-type SAMHD1 gene compared to 30% in the PTCL group with mutated SAMHD1 gene (p=0.01, Mann-Whitney U test), thus suggesting that alterations of SAMHD1 gene may represent a mechanism of SAMHD1 protein downregulation in a subset of PTCL. SAMHD1 expression or gene alterations did not correlate with FFP or OS in any PTCL histologic type, although the number of patients included in each group was not adequate to draw definite conclusions for prognostic significance. Conclusions: SAMHD1 gene mutations are frequently detected in a subset of PTCL and are associated with reduced expression of SAMHD1 protein. These findings reveal a novel mechanism (SAMHD1 mutations) of SAMHD1 downregulation in PTCL, and further support the tumor suppressor function of SAMHD1 gene in lymphomas. Disclosures Rosenquist Brandell: AbbVie: Honoraria; AstraZeneca: Honoraria; Illumina: Honoraria; Janssen: Honoraria; Roche: Honoraria.

To obtain insight into the ontogeny of mantle cell lymphoma (MCL), we assessed 206 patients from a morphological, immunohistochemical, and immunogenetic perspective. Our series included nodal ( n = 151), extranodal ( n = 28), and primary splenic ( n = 27) MCL cases. Skewing of the immunoglobulin heavy variable (IGHV) gene repertoire was noted, with only four IGHV genes accounting for 46% of cases and approximately 70% of cases (107/154) bearing an imprint of somatic hypermutation (SHM) ranging from minimal to pronounced. Interestingly, a distinctive immunophenotypic and immunogenetic profile was identified for primary splenic MCL, which was enriched for DBA.44-positive cases ( P P = 0.02) compared to nodal or extranodal cases, alluding to distinct immunopathogenetic and antigen selection processes. Expression of CD27 (considered a marker of activated B cells) was generally dissociated from SHM and was more prevalent in cases with no or minimal/borderline SHM. These findings support the idea that antigen drive is relevant for most MCL cases, although the specific antigens and the precise location of affinity maturation remain to be elucidated. Moreover, they raise the intriguing hypothesis of multiple cellular origins for MCL.

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.

Chronic lymphocytic leukemia patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinicobiological differences. Considering this, we assessed prognosis separately within mutated and unmutated chronic lymphocytic leukemia in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet-A mutated chronic lymphocytic leukemia 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 5- and 10-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 unmutated chronic lymphocytic leukemia patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-trearment (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 chronic lymphocytic leukemia 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 chronic lymphocytic leukemia.

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. De två första författarna delar förstaförfattarskapet.

Purpose: The role of antigen(s) in shaping the T-cell repertoire in chronic lymphocytic leukemia, although relevant for understanding malignant cell interactions with cognate T cells, is largely unexplored. Experimental Design: Here we profiled the T-cell receptor β chain gene repertoire in 58 chronic lymphocytic leukemia patients, focusing on cases assigned to well-characterized subsets with stereotyped clonotypic B-cell receptor immunoglobulins, therefore those cases most evidently selected by antigen (subsets #1, #2, and #4). Results: Remarkable repertoire skewing and oligoclonality were observed, and differences between subsets were noted regarding both T-cell receptor β chain gene usage and the extent of clonality, with subset #2 being the least oligoclonal. Longitudinal analysis of subset #4 cases revealed that although the repertoire may fluctuate over time, certain clonotypes persist, thus alluding to persistent antigenic stimulation. Shared (“stereotyped”) clonotypes were found between different patients, reflecting selection by common antigenic elements. Cross-comparison of our dataset with public databases showed that some T-cell clonotypes may have expanded secondary to common viral infections; however, the majority of clonotypes proved to be disease-specific. Conclusions: Overall, the T-cell receptor β chain repertoire in chronic lymphocytic leukemia is likely shaped by antigen selection and the implicated antigenic elements may concern epitopes that also select the malignant B-cell progenitors or, more intriguingly, chronic lymphocytic leukemia–derived epitopes. Clin Cancer Res; 22(1); 167–74. ©2015 AACR.

Stereotyped subset #2 (IGHV3-21/IGLV3-21) is the largest subset in CLL (~3% of all patients). Membership in subset #2 is clinically relevant since these patients experience an aggressive disease irrespective of the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene. Low-throughput evidence suggests that stereotyped subset #169, a minor CLL subset (~0.2% of all CLL), resembles subset #2 at the immunogenetic level. More specifically: (i) the clonotypic heavy chain (HC) of subset #169 is encoded by the IGHV3-48 gene which is closely related to the IGHV3-21 gene; (ii) both subsets carry VH CDR3s comprising 9-amino acids (aa) with a conserved aspartic acid (D) at VH CDR3 position 3; (iii) both subsets bear light chains (LC) encoded by the IGLV3-21 gene with a restricted VL CDR3; and, (iv) both subsets have borderline SHM status. Here we comprehensively assessed the ontogenetic relationship between CLL subsets #2 and #169 by analyzing their immunogenetic signatures. Utilizing next-generation sequencing (NGS) we studied the HC and LC gene rearrangements of 6 subset #169 patients and 20 subset #2 cases. In brief, IGHV-IGHD-IGHJ and IGLV-IGLJ gene rearrangements were RT-PCR amplified using subgroup-specific leader primers as well as IGHJ and IGLC primers, respectively. Libraries were sequenced on the MiSeq Illumina instrument. IG sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis conducted using an in-house, validated bioinformatics pipeline. Rearrangements with identical CDR3 aa sequences were herein defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. For the HC analysis of subset #169, we obtained 894,849 productive sequences (mean: 127,836, range: 87,509-208,019). On average, each analyzed sample carried 54 clonotypes (range: 44-68); the dominant clonotype had a mean frequency of 99.1% (range: 98.8-99.2%) and displayed considerable intraclonal heterogeneity with a mean of 2,641 subclones/sample (range: 1,566-6,533). For the LCs of subset #169, we obtained 2,096,728 productive sequences (mean: 299,533, range: 186,637-389,258). LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean: 148, range: 110-205); the dominant clonotype had a mean frequency of 98.1% (range: 97.2-98.6%). Intraclonal heterogeneity was also observed in the LCs, with a mean of 6,325 subclones/sample (range: 4,651-11,444), hence more pronounced than in their partner HCs. Viewing each of the cumulative VH and VL CDR3 sequence datasets as a single entity branching through diversification enabled the identification of common sequences. In particular, 2 VH clonotypes were present in 3/6 cases, while a single VL clonotype was present in all 6 cases, albeit at varying frequencies; interestingly, this VL CDR3 sequence was also detected in all subset #2 cases, underscoring the molecular similarities between the two subsets. Focusing on SHM, the following observations were made: (i) the frequent 3-nucleotide (AGT) deletion evidenced in the VH CDR2 of subset #2 (leading to the deletion of one of 5 consecutive serine residues) was also detected in all subset #169 cases at subclonal level (average: 6% per sample, range: 0.1-10.8%); of note, the 5-serine stretch is also present in the germline VH CDR2 of the IGHV3-48 gene; (ii) the R-to-G substitution at the VL-CL linker, a ubiquitous SHM in subset #2 and previously reported as critical for IG self-association leading to cell autonomous signaling in this subset, was present in all subset #169 samples as a clonal event with a mean frequency of 98.3%; and, finally, (iii) the S-to-G substitution at position 6 of the VL CDR3, present in all subset #2 cases (mean : 44.2% ,range: 6.3-87%), was also found in all #169 samples, representing a clonal event in 1 case (97.2% of all clonotypes) and a subclonal event in the remaining 5 cases (mean: 0.6%, range: 0.4-1.1%). In conclusion, the present high-throughput sequencing data cements the immunogenetic relatedness of CLL stereotyped subsets #2 and #169, further highlighting the role of antigen selection throughout their natural history. These findings also argue for a similar pathophysiology for these subsets that could also be reflected in a similar clonal behavior, with implications for risk stratification. Disclosures Sutton: Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.

The circulating tumor cells of chronic lymphocytic leukemia (CLL) are characterized by variably low surface IgM (sIgM) levels and signaling capacity, consequent to stimulation by tissue-based antigen. The variability is evident in both CLL subsets with unmutated and mutated immunoglobulin genes and affects tumor cell survival and proliferation. This appears clinically important because CLL with relatively higher sIgM levels/signaling capacity progress more rapidly than those with lower sIgM levels/signaling capacity (D'Avola, Blood 2016), likely due to a larger proliferative component at tissue sites. B-cell receptor (BCR)-signaling inhibitor ibrutinib exerts its therapeutic activity in CLL by irreversibly binding the Bruton's tyrosine kinase (BTK) at Cys481 in the active site. This leads to redistribution of CLL cells from tissue sites into peripheral blood and to a selective recovery of sIgM levels and function, consequent to release from antigen drive at tissue sites (Drennan, Clin Cancer Res 2019). We have now investigated the hypothesis that sIgM levels on the CLL cells affect duration of response to ibrutinib therapy possibly consequent to circumvention of the pharmacological BTK inhibition. Seventy CLL patients participating in the "real-world" observational study at the University of Southampton (NIHR/UKCRN ID: 31076) were investigated for phenotypic, molecular and functional characteristics associated with response to single-agent ibrutinib. Time to progression requiring a new treatment from ibrutinib start (TTNT) was used as primary endpoint to measure duration of response. The levels of sIgM were measured by flow cytometry. Anti-IgM induced signaling was determined by immunoblotting or iCa2+ mobilization. BTK and PLCγ2 mutations were determined using the Illumina NexteraXT kit and a MiSeq at the University of Southampton and validated independently at Karolinska Institutet. Our previous cut-offs of 50 (MFI) or 5 (iCa2+ % mobilization) were used to distinguish patients with high/low sIgM levels or signaling capacity, respectively. Median follow-up from ibrutinib start was 42 months (range 12-70). Pre-ibrutinib sIgM levels (range 7-618, median 65; high sIgM MFI 46/70, 66%) and sIgM signaling capacity (range 1-98; median 45; high sIgM signalers 56/70, 80%) were broadly heterogeneous. However, median values were significantly higher than the general CLL population at diagnosis, as expected in progressive CLL. By univariate analyses of clinical, phenotypic, FISH and immunogenetic characteristics for TTNT, high sIgM was the only parameter predicting shorter duration of response to ibrutinib (p=0.02). Only 2/24 CLL with low sIgM levels (CLLIgM_lo) had progressed (median TTNT not reached) compared to 15/46 CLL with high sIgM (CLLIgM_hi, 14% at 12 months, 18% at 24 months, 34% at 36 months, 44% at 42 months) during therapy. The levels of sIgM correlated significantly with sIgM signaling capacity prior to treatment (r= 0.68; p From in vitro analysis of 13 therapy-naive CLL samples, the degree of inhibition by ibrutinib of anti-IgM induced signals downstream to BTK correlated negatively with sIgM levels (r=-0.68,p=0.01) and signaling (r=-0.71,p=0.009). Anti-IgM induced signals were then measured in 12 patients (7 CLLIgM_hi, 5 CLLIgM_lo) after 1, 4 and 12 weeks of ibrutinib therapy. These patients had not acquired BTK or PLCγ2 mutations and BTK phosphorylation appeared completely inhibited at all time-points. Mean anti-IgM induced iCa2+ mobilization and ERK1/2 phosphorylation were significantly reduced but not abolished during therapy and degree of inhibition was variable between individuals. Remarkably, degree of inhibition of anti-IgM-induced iCa2+ mobilization and pERK1/2 was significantly lower in CLLIgM_hi than in CLLIgM_lo (p These results confirm that sIgM signaling is dependent on sIgM levels and that it can circumvent BTK blockade when sIgM levels are high. They suggest that high sIgM signaling can drive ibrutinib resistance despite ability of ibrutinib to fully occupy the BTK phosphorylation pocket. A possibility is that those CLL cells with high sIgM represent a potentially dangerous subpopulation equipped to migrate to tissue and to receive proliferative stimuli. These cells might be targeted by a combinatorial therapeutic approach with ibrutinib. Disclosures Johnson: Takeda: Honoraria; Kite: Honoraria; Bristol-Myers Squibb: Honoraria; Genmab: Honoraria; Incyte: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Epizyme: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria. Duncombe:Abbvie: Other: Advisory Board membership;Educational support; Gilead: Other: Advisory Board membership; Janssen: Other: Advisory Board membership;Educational support; Novartis: Other: Advisory Board membership. Scarfo:Janssen: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria. Sutton:Gilead: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Ghia:Pharmacyclics LLC, an AbbVie Company: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding; ArQule: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Acerta/AstraZeneca: Consultancy, Honoraria; Novartis: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding. Forconi:Roche: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Menarini: Consultancy; Novartis: Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Gilead Sciences: Research Funding.

One of the most common secondary resistance mechanisms to ibrutinib (IBR, a first-generation, irreversible Bruton's tyrosine kinase [BTK] inhibitor) in CLL is the development of mutations in BTK involving Cys481, leading to impaired drug binding (Woyach et al, NEJM 2014; Furman et al, NEJM 2014). The mechanisms of resistance to second generation BTK inhibitors are currently unknown. We aimed to assess the spectrum of acquired BTK mutations in patients with CLL progression on zanubrutinib (ZANU), a second-generation, irreversible inhibitor of BTK. We identified 38 CLL patients, treated with ZANU on clinical trials (NCT02343120, NCT02569476, NCT03336333, NCT02795182) at three centres, for whom serial samples were available. Four of 38 patients (10.5%) had CLL progression on ZANU (time to progression 5, 26, 29 and 48 months) and underwent amplicon next generation sequencing (NGS) of BTK (exon 11, 15, 16) and PLCG2 (exon 16, 19-20, 24, 27-28). Remarkably, we detected a BTK kinase domain mutation, BTK Leu528Trp (NM_000061.2:c.1583T>G), in all four patients progressing on ZANU. In addition, all four patients had detectable Cys481 mutations at lower variant allele frequency (VAF) than the BTK Leu528Trp (median BTK Leu528Trp 34.9% vs BTK Cys481 9.1%). Analysis of sequence reads from amplicon NGS and RNA-sequencing data demonstrated that BTK Leu528Trp and BTK Cys481 mutations were present on different alleles. Assessment of the BTK Leu528Trp and BTK Cys481 mutations with high sensitivity droplet digital PCR (ddPCR) confirmed the absence of both mutations prior to ZANU exposure in all patients (sensitivity 0.1% VAF). Longitudinal analysis of the four patients with the BTK Leu528Trp mutation demonstrated the appearance of the Leu528Trp coincident with rising measurable disease and subsequent clinical CLL progression. We then went on to test patients on ZANU without disease progression but with persistent measurable disease (n=34) by ddPCR and detected three further patients harbouring low level BTK Leu528Trp mutations (VAF The BTK Leu528Trp mutation has been described only once previously in a patient in the context of IBR resistance (who transformed with Richter's syndrome) where it co-occurred with Cys481 mutations (Maddocks et al, JAMA Oncol 2015). As the prevalence of BTK Leu528Trp among progressive disease samples in our cohort exceeds all prior reports in IBR-treated patients, we sought to further understand the specificity of BTK Leu528Trp for ZANU progression. Targeted sequencing in a cohort of 49 patients progressing on IBR from the European Research Initiative on CLL (ERIC) did not detect the BTK Leu528Trp in any patients (sensitivity 1% VAF). We went on to perform biochemical and cellular studies on the BTK Leu528Trp mutation. Assessment of enzymatic activity of BTKLeu528Trp demonstrated a significant loss of activity compared to both BTKWT and BTKCys481Ser. This was further confirmed by assessing BTK autophosphorylation in HEK293 cells. Autophosphorylation at BTK Tyr223 was markedly reduced in HEK293 cells stably expressing BTKLeu528Trp compared to both BTKCys481Ser and BTKWT. In addition, a crystal structure of apo-BTKLeu528Trp was solved to understand effects of BTKLeu528Trp on ZANU binding to BTK. The alignment of the crystal structure of apo-BTKLeu528Trp with that of BTKWT-ZANU or the modeled structure of BTK-ATP suggested potential steric clashes between BTKLeu528Trp and ZANU (Figure 1A), as well as BTKLeu528Trp and ATP (Figure 1B). In conclusion, we have described the novel enrichment of BTK Leu528Trp mutations occurring in patients with CLL progressing on ZANU and both structural and experimental data consistent with this mutation resulting in interference with both ATP and ZANU binding to BTK. These findings emphasize the potential for agent-specific resistance mutations with second generation BTK inhibitors and the need to include these mutations in diagnostic screening for BTK resistance in the clinic. SH/CPST co-first authors, CT/PB co-senior authors Disclosures Handunnetti: Abbvie: Other: Travel Grant; Gilead: Honoraria. Zhou:Beigene: Employment. Sun:Beigene: Employment. Xing:Beigene: Employment. Zhang:Beigene: Employment. Guo:Beigene: Employment. Sutton:Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Ghia:Dynamo: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy; Novartis: Research Funding; Juno/Celgene: Consultancy, Honoraria. Scarfo:AstraZeneca: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Seymour:Takeda: Consultancy; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; Janssen: Research Funding; BeiGene: Research Funding. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Liu:Beigene: Employment. Cheah:Roche, Janssen, MSD, Gilead, Loxo Oncology, Acerta, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Abbvie: Research Funding; Roche: Other: Travel expenses. Tam:Janssen: Honoraria, Research Funding; BeiGene: Honoraria; Pharmacyclics LLC, an AbbVie company: Honoraria; Roche: Honoraria; Novartis: Honoraria; AbbVie: Honoraria, Research Funding. Blombery:Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy.

The overall survival (OS) of patients with chronic lymphocytic leukemia (CLL) has improved over the last decades mainly due to advances in the understanding of the disease biology and the introduction of novel therapeutic approaches(1). In the present retrospective study we investigated trends in OS in subgroups of cases defined by genetic and immunogenetic features aiming at addressing the question whether advances in chemoimmunotherapy had a uniform impact across all CLL patients. We found that such advances have translated into prolonged OS in all prognostic subgroups examined except those carrying TP53 abnormalities, as expected, but also those assigned to stereotyped subsets #1 and #2, that are generally devoid of such gene aberrations. This latter finding, reported here for the first time, indicates the need for alternative treatment options for these patients.

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.