Abstract: Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

Abstract: Introduction. The majority of SMZLs display an indolent course, however the disease is still incurable and a significant proportion of patients (~25-30%) experience poor outcomes surviving 〈 5 years. Molecular alterations of SMZL are promising biomarkers, which might improve risk stratification of patients. The main objective of the study is to test the impact of molecular aspects on disease-specific survival prognostication in newly diagnosed SMZL. Methods. IELSG46 is a multicenter, international, retrospective, observational study in which already existing and coded health-related personal and biological material is used. The study included adults, who received a diagnosis of SMZL on spleen histology with a follow up 〉 5 years, and for whom tumor material collected before initiation of medical therapy was available. Mutation analysis was performed by CAPP-seq targeted deep next generation sequencing of tumor genomic DNA. A stringent bioinformatic pipeline was applied to suppress the background noise allowing to call variants with a sensitivity of 5x10-2 in FFPE derived DNA. Copy number variations (CNVs) were identified by using the sequencing reads-based GATK4-CNV algorithm. IGHV rearrangements were obtained by using LymphoTrack® IGH FR1 Assay Panel kit. Molecular clusters were identified by an iterative algorithm that maximizes genetic distinctiveness of subgroups by reassigning patients between clusters that are created a priori based on the co-occurrence of genetic lesions. Relative survival, defined as the ratio between actuarial survival observed in the SMZL cohort and expected survival of the general population matched to patients by geographical origin, sex, age and calendar year of diagnosis, was calculated using the Ederer II method. Results. The analysis included 303 patients with a SMZL diagnosis confirmed on spleen histology. The sample size allowed to identify 30% differences in survival for molecular subgroups comprising at least 5% of cases with a statistical power between 80-100%. Median follow-up was 9.2 years. At 10 years, 85% of patients were alive, consistent with the known indolent behavior of this lymphoma. Genes recurrently affected by non-synonymous somatic mutations in 〉 10% of SMZL included KLF2 (24%), NOTCH2 (19%), KMT2D (15%), TNFAIP3 (13%), EP300 and TP53 (10%). Deletion 7q was documented in 25% of cases and IGHV1-2*04 usage in 32%. By cluster analysis, three major molecular subgroups were identified, each of them characterized by a NOTCH pathway mutated gene (Fig. 1A). The first cluster was defined by NOTCH2 and/or KLF2 mutations and was enriched in TNFAIP3 mutations and IGHV1-2*04 gene usage (Fig. 1A). The second cluster was defined by SPEN mutations, and was enriched in KMT2D and other epigenetic gene mutations (Fig. 1A). The third cluster was enriched in NOTCH1 mutations (Fig. 1A). By relative survival analysis, the NOTCH2/KLF2 cluster showed a lower survival compared to the matched general population, indicating a significant impact of the disease on patients' expected survival (Fig. 1B). Conclusions. The large sample size and inclusion of SMZL confirmed by spleen histopathology review allowed for precise estimation of the prevalence of KLF2 and NOTCH2 mutations in this lymphoma. Three molecular clusters were identified in SMZL, each of them containing a NOTCH pathway gene, supporting the relevance of NOTCH signaling in the pathogenesis of SMZL. Patients belonging to the NOTCH2/KLF2 cluster had a lower relative survival compared to the matched general population. Disclosures Traverse-Glehen: Astra Zeneca: Other: Travel; Takeda: Research Funding. Gomes da Silva:Roche: Other: Institution's payment for consultancy, Travelling support; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Celgene: Other: Travelling support; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Institution's payment for consultancy, Travelling support; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Research Funding. Ladetto:Celgene: Honoraria; Jannsen: Honoraria; Acerta: Honoraria; Abbvie: Honoraria; Sandoz: Honoraria; Roche: Honoraria. Rambaldi:Pfizer: Consultancy; Novartis: Consultancy; Omeros: Consultancy; Italfarmaco: Consultancy; Amgen Inc.: Consultancy; Roche: Consultancy; Celgene: Consultancy. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Zinzani:MSD: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau. Gaidano:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Salles:Servier: Honoraria; Abbvie: Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Amgen: Honoraria; BMS: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board, Research Funding; Acerta: Honoraria; Janssen: Honoraria, Other: Advisory Board; Merck: Honoraria; Pfizer: Honoraria; Morphosys: Honoraria; Gilead: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Servier: Honoraria, Other: Advisory Board; Takeda: Honoraria. Zucca:Celltrion: Consultancy; AstraZeneca: Consultancy.

Abstract: Introduction: Despite the improvements, still too many patients die for their lymphomas and novel compounds are needed. We present a new small molecule, EG-011 (PCT/EP2018/057678), with in vitro and in vivo anti-cancer activity in lymphoma models. Methods: Lymphoma and solid tumor cell lines were exposed to a large range of concentrations of EG-011 as single agent for 72h, followed by MTT proliferation assay and IC50 calculation. Cell viability of twelve acute lymphoblastic leukemia (ALL) primary patient cells from different high-risk subgroups (VNN2+, E2A-HLF, refractory T and IKZF plus) co-culture with marrow-derived MSCs were assayed after 72h of incubation with EG-011 and controls. Apoptosis assay was measured with annexin V by FACS. Xenografts were established s.c. into the left flanks of female NOD-SCID mice; treatment (200 mg/kg, i.p. 5 days per week) started with already established tumors. Combinations were evaluated with Chou-Talalay combination index (CI): synergism ( & lt;0.9), additive (0.9-1.1), antagonism/no benefit ( & gt; 1.1) after 72 hr treatments. Results: EG-011 presented a median IC50 of 2.25 μM in 62 lymphoma cell lines (95% C.I. 1-5μM). A higher activity was observed in a group of 21 cell lines that had a median IC50 of 250 nM (95% C.I. 40-600 nM). Among these there were 11 germinal center B cell (GCB) diffuse large B cell lymphomas (DLBCL) (sensitive n=11/21, resistant n=9/41, P & lt; 0.05), 4 mantle cell lymphoma (MCL) (sensitive n=4/21, resistant n=6/41, P n.s.), 3 marginal zone lymphoma (sensitive n=3/21, resistant n=2/41, P n.s.). EG-011 did not show any anti-proliferative activity in a panel of 25 solid tumor cell lines (IC50s & gt; 10 μM), Among 12 primary ALL samples, 7 were sensitive to EG-011 with IC50 values between 0.3-4.6 µM after 72h, 5 displayed IC50 higher than 20 µM. A dose-dependent increase in cell death (20-55%) was observed in lymphoma cell lines (OCI-LY-19 and REC1) (500 nM and 2 μM; 72h). No cytotoxicity was seen in PBMCs from two healthy donors after treatment at 1 and 10 μM for 24h and 48h. In an in vivo xenograft experiment with the MCL REC-1 cell line, EG-011 delayed tumor growth (Day 6, Day 7, Day 9, P & lt; 0.05) and tumor weight. EG-011-treated tumors were 2.2-fold smaller than controls (P & lt; 0.001). Combinations were tested in DLBCL (OCI-LY-1, OCI-LY-8, TMD8) and MCL (REC1, MINO). EG-011 was synergistic with rituximab, bendamustine, venetoclax, ibrutinib and lenalidomide in all tested cell lines. Conclusion: The selective anti-lymphoma activity, in both in vitro and in vivo models, and the observed in vitro synergisms with FDA approved targeted agents make EG-011 a novel intriguing new drug candidate deserving further preclinical studies. Citation Format: Eugenio Gaudio, Filippo Spriano, Chiara Tarantelli, Matilde Guala, Eugenia Riveiro, Gaetanina Golino, Antonio Lupia, Giosuè Costa, Roberta Rocca, Luciano Cascione, Silvia Jenni, Yi-Chien Tsai, Beat Bornhauser, Stefano Alcaro, Francesco Paduano, Francesco Trapasso, Emanuele Zucca, Anastasios Stathis, Natalina Pazzi, Franco Cavalli, Francesco Bertoni. EG-011 is a novel small molecule with in vitro and in vivo anti-tumor activity against lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4796.

Abstract: Deletion of 17p ( TP53 ) identifies a rare subset of chronic lymphocytic leukaemia (17p‐ CLL) with aggressive behaviour. Genome‐wide DNA‐profiling was performed to investigate 18 patients with 17p‐ CLL. All cases had multiple copy‐number (CN) changes. Among the several recurrent CN changes identified, 8q24.13‐q24.1‐gain ( MYC ), 8p‐loss ( TNFRSF10A/B , also known as TRAIL1/2 ) and 2p16.1‐p14‐gain ( REL/BCL11A ) appeared frequently represented. 8p‐loss and 2p16.1‐p14‐gain also appeared clinically relevant and predicted significant shorter time from diagnosis to treatment (8p‐loss) and overall survival (8p‐loss and 2p16.1‐p14‐gain, P   〈  0·05). These observations document a highly unstable genome in 17p‐ CLL and suggest that additional genes outside the TP53 locus may be important for tumour behaviour.

Abstract: Introduction. Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a minimal residual disease (MRD) positive partial remission (PR) which is maintained until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. The study aims at characterizing the adaptation process allowing residual CLL cells to persist despite BTK inhibition. Methods. The IOSI-EMA-001 study (NCT02827617) is an observational study consisting in the prospective and longitudinal collection of peripheral blood samples and clinical data from high risk CLL patients treated with ibrutinib. Peripheral blood CLL cells longitudinally drawn from patients before treatment start and at fixed timepoints under ibrutinib were monitored by: i) next generation flow cytometry approaches for changes in proliferation rate, surfaceome, and pathway activation; and ii) CAPP-seq targeted deep next generation (sensitivity ~10-3) for clonal evolution. Results. The study cohort comprised 31 high risk CLL patients, including 15 treatment naïve, 16 relapsed, 80% IGHV unmutated, 42% 17p deleted and 55% TP53 mutated. Median duration of ibrutinib treatment was 45 weeks (24-72 weeks). All patients obtained a MRD positive PR that was maintained in all but one who progressed with a PLCG2 mutation (VAF 3%). Compared to baseline, under ibrutinib therapy CLL cells slowed down their proliferation, as suggested by the decreased expression of Ki-67, the reduction of the proliferating fraction (CXCR4dimCD5bright), and the increase of the resting fraction (CXCR4brightCD5dim). Compared to baseline, under ibrutinib therapy CLL cells also upregulated BCR and adhesion/homing proteins, and decreased the expression of BCR inhibitor proteins. Upon stimulation of the BCR with anti-IgM, the downstream path through pBTK and pPLCG2 was inhibited by ibrutinib, while conversely the downstream path through pAKT and pERK was still inducible throughout all the assessed timepoints. The proportion of CLL cells harboring nuclear localization of NF-kB progressively increased over time under ibrutinib. NF-kB nuclear localization was inducible throughout all the assessed timepoints by CD40L stimulation of the non-canonical NF-kB pathway, but not by anti-IgM stimulation of the BCR/canonical NF-kB pathway. Overall, 880 individual mutations were longitudinally discovered and monitored across a total of 121 sequential timepoints collected during ibrutinib treatment. Clonal evolution was observed in (67.7%) cases, a proportion rate previously documented in CLL treated with chemoimmunotherapy. Clonal evolution appeared to be heterogeneous involving different genes without a stereotypic targeting. Consistently, none of the main driver gene mutations was homogeneously selected or suppressed by ibrutinib suggesting that the biological adaptation of CLL cells under ibrutinib is not genetically driven. Clonal evolution propensity was not associated with any of the biomarkers of the disease, and it did not decrease over time under ibrutinib. Conclusions. Taken together these results suggest that residual CLL cells persisting under ibrutinib therapy adapt their phenotype by upregulating adhesion molecules, chemokine receptors and BCR molecules, and by maintaining a competence of BCR signaling through the PI3K/AKT/ERK pathway. The progressive selection of CLL cells having NF-kB in the nucleus, likely due to the BTK independent non-canonical NF-kB pathway, might explain their survival despite ibrutinib therapy. Finally, clonal evolution is not suppressed by ibrutinib chemotherapy, and despite does not seem to be directly involved in such adaptation process, may ultimately favor the acquisition of BTK and PLCG2 ibrutinib resistance mutations. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Montillo:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Tedeschi:Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; AbbVie: Consultancy. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria.

Abstract: Hairy cell leukemia (HCL) is a rare B-cell neoplasm with largely unravelled features of lymphomagenesis. Molecular mechanisms of disease are largely unknown and genomic aberrations have infrequently been investigated in HCL. In order to elucidate the genetic mechanisms of HCL pathogenesis, we analyzed copy number changes and loss of heterozigosity (LOH) by genome wide DNA profiling with the Affymetrix Human Mapping 250k Nsp arrays in 16 HCL. All investigated cases expressed multiple pre- (IgM+/−D) and post-switch (IgG+/−A) isotypes. Tumor IGHV gene transcript homology to germline ranged from 88.4 to 98.6%, with 4/16 HCL having 〉 98% homology. Four of 16 patients were refractory or had progressive disease after first line purine-analogue-based treatments. CD103+ve hairy cells were 〉 60% in all test samples. Genome wide DNA profiling demonstrated gross non recurrent copy number deletions in only 4/16 cases (25%), affecting 14q24-q32, 3q23, 3q27.3-q29, 8p12-pter 10q21-q23, 10q25-q26, 17p11-pter, 17q11-q21, 17q22–23, Xp21.2-p22.11, Xq13.3, Xq21.1-q21.33, Xq22.2–q28, the whole chromosomes 21 and X. Five of 16 patients (31%) presented LOH. Most interestingly, the 5 patients showed LOH regions, with a region of overlap in two patients at 6q25.1, without any copy number changes, indicative of uniparental disomy. Genes involved in bone marrow fibrosis, multiple isotype expression and response to treatment were specifically investigated. Interestingly, among genes critical for fibrosis, FGF12, a member of the fibroblast growth factors family, was specifically targeted by a complex chromosomal rearrangement at 3q27.3-q29, and reverse transcript-polymerase chain reaction confirmed FGF12 transcript expression. Among genes associated with poor response, p53 deletion was documented in a non-responder HCL. Conversely, no abnormalities at 14q32 locus (duplications or gains) were observed at IgH 14q32 region in these multiple IgH isotype expressing HCL. Overall, our analysis showed a highly stable genome with lack of gross recurrent abnormalities, which is an additional unique feature of HCL among B-cell neoplasms. However, our genomic analyses were able to identify specific chromosomal rearrangements targeting genes involved in fibrosis. Lack of gains of 14q32 locus was consistent with our hypothesis that multiple IgH isotypes are generated at RNA level in multiple IgH expressing HCL. The present analysis provides the basis for further molecular and epigenetic investigations.

Abstract: Chronic lymphocytic leukemia (CLL) with deletion of 17p(p53) locus (17p- CLL) identifies the most aggressive CLL subset, due to active disease course and resistance to current treatments. Intrinsic mechanisms of the poor prognosis of 17p- CLL are scarcely known. In order to identify additional lesions occurring in 17p- CLL which contribute to prognosis, we performed genome-wide DNA profiling in 18 cases of 17p- CLL (median follow-up 25 months, range 4–107), using high-resolution single-nucleotide polymorphism (SNP) arrays (Affymetrix Human Mapping Nsp 250 k arrays). Chromosomal 11q, 13q or 17p deletions and trisomy 12 were determined by FISH. Tumor IGHV usage and mutational status were determined by PCR/sequencing of the tumor gDNA/cDNA. Median interval from diagnosis to progression (11 months, p=0.000122) and treatment (16 months, p=0.000827) were representative of the bad behavior of our 17p- CLL cases if compared to 95 non17p- CLL patients from our internal cohort (68 and 69 months, respectively). Interestingly, 3/18 (16%) 17p- CLL expressed the IGHV3–74 segment which is rare in normal B-cells (1,4%, p=0.02) and in non17p- CLL (2.1%, p=0.02). Tumor IGHV genes were unmutated in 10/18 and mutated in 8/18 17p- CLL. All cases carried 17p13.1 deletion in more than 60% nuclei, as assessed by FISH. Genome-wide DNA profiling was 99% concordant with FISH (71/72 data-points; one 13q deletion detected by FISH in 20% of the nuclei was missed by the microarray) and confirmed 17p deletion in all cases. Deletions at the 13q14.3 locus recurred in 50% cases. Only one case had an extra-copy of chromosome 12. No cases carried ATM deletion. Other recurrent lesions identified by genome-wide DNA profiling were: gains of 2p16.1-pter (30%), 3q24-qter (20%), 8q23.3-qter (30%), 17q21.2-q22 (30%), 14q32 (30%); losses of 8p12-pter (30%), 9q21.33-q22.1 (20%), 14q32 (40%), 20p12.1-pter (20%). Approximately half of the cases had concomitant losses of 8p or 17p and gains of the 8q or 17q regions, respectively, suggesting the presence of isochromosomes 8 or 17. Indeed, isochromosome 17 was evident in one case analyzed by conventional cytogenetics. Six patients had one or more regions of loss of heterozygosity (LOH) longer than 5 Mb in the absence of copy number reduction, suggestive of uniparental disomy. Several lesions, such as those involving chromosomes 3, 8 and 9, associate with poor clinical behavior in other B-cell tumors. Thus, we investigated the prognostic significance of the lesions identified in our series. Despite the small number of cases, we found that 17p- CLLs with loss of 8p12-pter, which contains tumor necrosis factor and defensin family genes, associated with worse treatment free interval from diagnosis (12 vs 46 months in 8p- vs non 8p- CLL, p=0.04). Overall, our analysis identified new recurrent genomic abnormalities at high frequencies that may specifically contribute to a different biologic and clinical behavior within 17p- CLL. Among these, isochromosomes 8 and 17 were particularly common, and 8p loss might identify a new aggressive subentity within 17p- CLL.

Abstract: A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B‐cell receptors (BCR), i.e. characterized by non‐random combinations of immunoglobulin heavy‐chain variable ( IGHV ) genes and heavy‐chain complementarity determining region‐3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such ‘stereotyped’ BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time‐to‐treatment (TTT) and presence of known prognosticators. Sixty‐nine clusters (319 IG‐rearrangements, 22·4%) with stereotyped BCR were identified. Among 30 confirmed clusters (≥3 IG‐rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster‐biased amino acid changes were found throughout IGHV sequences of these ‘M clusters’. Regarding clinical outcome: (i) UM CLL from the IGHV1‐2/1‐3/1‐18/1‐46/7‐4‐1/IGKV1‐39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3‐21/IGLV3‐21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3‐21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.