Stereotyped Subsets Research Articles

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.

Exploiting B-cell Receptor Stereotypy to Design Tailored Immunotherapy in Chronic Lymphocytic Leukemia.

Approximately 30% of patients with chronic lymphocytic leukemia (CLL) can be grouped into subsets with stereotyped B-cell receptor immunoglobulin (BcR IG) displaying remarkable similarity in the heavy complementarity-determining region 3 (VH CDR3). Here, we investigated whether the consensus VH CDR3 sequences from CLL stereotyped subsets can be exploited for immunotherapy approaches. Immunogenic epitopes from the consensus VH CDR3 sequence of the clinically aggressive subsets #1 and #2 and from Eμ-TCL1 mice, which spontaneously develop CLL with BcR IG stereotypy, were identified and used to generate specific HLA class I- and II-restricted T cells in vitro. T-cell reactivity was assayed in vitro as IFNγ production. Bone marrow-derived dendritic cells loaded with the peptides were used as vaccination strategy to restrain leukemia development in the Eμ-TCL1 mouse model. These stereotyped epitopes were naturally processed and presented by CLL cells to the VH CDR3-specific T cells. Furthermore, we validated the efficacy of VH CDR3 peptide-based immunotherapy in the Eμ-TCL1 transplantable mouse model. Immunization of mice against defined VH CDR3 peptide epitopes, prior to the challenge with the corresponding leukemia cells, resulted in the control of CLL development in a significant fraction of mice, and increased overall survival. Our data highlight the immunogenicity of stereotyped VH CDR3 sequences and support the feasibility and efficacy of their use for novel cancer vaccine in CLL. Such approach has the advantage to generate "off-the-shelf" therapeutic vaccines for relevant groups of patients belonging to stereotyped subsets.See related commentary by Seiffert, p. 659.

The IGLV3-21R110 Defines a Subset of Chronic Lymphocytic Leukemia with Intermediate Epigenetic Subtype and Poor Outcome

Introduction: B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B-cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling conferring aggressive behavior. Epigenetic studies have defined three CLL subtypes based on methylation signatures reminiscent of pre- and post-germinal center B-cells named naïve-like (n-CLL), intermediate (i-CLL) and memory-like CLL (m-CLL) with different biological features. i-CLL carry a borderline IGHV mutational load and a significant higher usage of IGHV3-21/IGLV3-21. The integration of these factors might translate into novel insights in CLL pathogenesis with implications on the proposed stratification of the patients. Aim: To determine the clinical and biological features of the IGLV3-21R110 in CLL in the light of the epigenetic subtypes and immunogenetic, genomic and transcriptomic landscapes of the tumors. Methods: We characterized the immunoglobulin (IG) gene of 584 CLL cases from whole-genome/exome and RNA sequencing using our recently developed algorithm IgCaller (Nadeu et al., Nat. Commun. 2020) and MiXCR, respectively. The genomic makeup of the tumors was obtained from whole-genome/exome sequencing while RNA sequencing data for 369 cases was used for gene expression analyses. Expression levels of WNT5A and WNT5B were verified by quantitative PCR with reverse transcriptase. Primary end points were time to first treatment (TTFT) and overall survival (OS) calculated from the date of diagnosis. All patients gave written informed consent. The study was approved by the Ethics Committee of the Hospital Clínic of Barcelona. Results: The IGLV3-21R110 was detected in 6.5% of cases being similarly distributed between mutated (6.5%) and unmutated (6.6%) IGHV cases (P=0.56). In contrast, the IGLV3-21R110 was found in 30/79 (38%) i-CLL compared to only 5/291 (1.7%) m-CLL and 1/189 (0.5%) n-CLL (P<0.001). All stereotyped subset #2 cases carried IGLV3-21R110 while 62% of IGLV3-21R110 i-CLL had non-stereotyped IG genes. IGLV3-21R110 i-CLL had a borderline IGHV mutational status (median 97.7%) that was higher than i-CLL lacking the IGLV3-21R110 (median 96.2%, P=0.005). IGLV3-21R110 i-CLL had significantly higher number of SF3B1 and ATM mutations, and total number of driver alterations. Nonetheless, the R110 mutation was the sole alteration in one i-CLL case and accompanied only by del(13q) in three. Although composite regarding IGHV mutational status, IGLV3-21R110 i-CLL transcriptomically resembled naïve-like/unmutated IGHV CLL and had a specific expression signature of 64 genes with overexpression of WNT5A and WNT5B as hallmarks. No differences were observed in the expression profile of subset #2 and non-subset #2 IGLV3-21R110 i-CLL tumors. On the other hand, i-CLL lacking the IGLV3-21R110 phenotypically mirrored memory-like/mutated IGHV cases. In relation to prognosis, IGLV3-21R110 i-CLL had a short TTFT and OS similar to n-CLL/unmutated IGHV cases whereas non-IGLV3-21R110 i-CLL had a good prognosis similar to memory-like/mutated IGHV. Therefore, i-CLL cases, which have been associated with an intermediate prognosis between m-CLL and n-CLL in previous studies, can be divided in two subgroups of cases with opposed clinical evolutions based on the IGLV3-21R110. Indeed, the IGLV3-21R110 and n-CLL subtype retained independent prognostic value in multivariate analyses while the i-CLL lost its prognostic prediction both for TTFT and OS. The prognostic value of the IGLV3-21R110 was also independent of the IGHV mutational status. In terms of applicability in the clinics, all n-CLL cases were classified as unmutated IGHV and 98% of m-CLL were mutated IGHV. Thus, either a complete IG characterization (IGHV mutational status and IGLV3-21R110) or the integration of the n-CLL subtype and IGLV3-21R110 identified virtually the same subset of patients with aggressive disease. Conclusions: The IGLV3-21R110 defines a CLL subset with borderline IGHV mutations, specific driver alterations, a gene expression signature including WNT5A/B overexpression, and an unfavorable prognosis independent of the IGHV mutational status and epigenetic subtypes. Our findings support the identification of IGLV3-21R110 CLL as a particular subgroup of the disease with relevance in the risk stratification of the patients. Disclosures Nadeu: Janssen: Honoraria. Campo:NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America..

Targeted Next Generation Sequencing Improves Diagnosis in Unclassifiable Leukemic Indolent B-Cell Non-Hodgkin Lymphoma and Identifies a Subset with Recurrent MYD88 Mutations in a Prospective Multicentre Study

Introduction: Patients with leukemic indolent B-cell Non-Hodgkin Lymphomas (LI B-NHL) that do not easily fall into a distinct pathological category with standard diagnostics can pose a challenge as their optimal management is uncertain. Lack of a clear diagnostic label can deny patients access to novel therapies as regulatory approval of drugs is largely granted within histological categories. Moreover, these patients may be excluded from participation in clinical trials. We report findings from a prospective study evaluating targeted next generation sequencing (NGS) of circulating malignant B-cells in this setting. Methods: Over a period of 4 years, 108 patients with LI B-NHL, deemed unclassifiable on standard peripheral blood (PB) diagnostics, were prospectively enrolled from 14 centres around the UK including ours. Patients with non-clonal lymphocytosis, confirmed CLL-type MBL, CLL (Matutes score 4 or 5), MCL, HCL, high grade lymphoma on standard PB diagnostics were excluded. Clinical and morphological characteristics were analysed. PB immunophenotyping was used for characterisation including ROR1 expression. CD15+ cells were positively selected with magnetic beads for germline DNA. Tumor-germline pairs were analysed with theEuroclonality-NGS DNA capture panel, and ARResT/Interrogate and complementary pipelines, to characterise clonal immunoglobulin rearrangements, translocations and somatic mutations (Stewart et al. ASH annual meeting 2019). Germline variants were subtracted to improve somatic mutation detection. Results: Median age was 72yrs with M:F ratio of 2:1. Incidental lymphocytosis on a routine blood count was the commonest mode of presentation with only a third of patients symptomatic at presentation. Median lymphocyte count was 12.4 x 109/L at the time of sampling. Just under 50% of cases had partial or complete CD5 expression, CD19 and 20 expression was moderate to strong in most cases while ROR1 was negative in most cases. NGS was able to assign a diagnostic category in 11/90 (12%) cases based on detection of IGH;CCND1 (6), IGH;BCL2 (2), IGH;BCL3 (2) translocations and in one case a BRAF mutation with confirmation of hairy cell leukaemia on immuno-morphology. These tests were not triggered by standard diagnostic pathways at initial presentation. Two novel translocations of uncertain significance were detected - RAD51B:BIRC3 and IGHMswitch-MICALL2/INTS1. Clonal IGHV-IGHD-IGHJ rearrangements were detected by NGS and/or Sanger sequencing with predominantly IGHV3 and IGHV4 gene usage, IGHV4-34 being the most common with 15 cases. The majority of rearrangements showed somatic hypermutations above 2% and none could be assigned to the 19 major CLL-associated stereotyped subsets (using ARResT/AssignSubsets). Somatic mutations were detected in 74/108 cases. The most frequently mutated genes were TP53 in 16/108 (15%) and the hotspot MYD88 L265P mutation in 14/108 (13%) with VAF of 20-46%. Other variants detected were distributed among genes associated with NFkB signalling and chromatin remodelling (Figure 1). The MYD88 L265P mutation was present in a fifth of cases with mutations detected (14/74) and was the sole change seen in 7 cases. Accompanying mutations in the remaining cases included CD79B (2), POT1 (1), NFKBIE (1). No concomitant CXCR4 mutations were detected. Median age of the MYD88 L265P cohort was 68 years with male predominance (M:F=10:4). 9/14 presented with an incidental lymphocytosis and median count of 11.18 x 109/L. Lymphadenopathy was present in 3/14 (21%) while 7/14 (50%) had splenomegaly. A paraprotein was detected in 6/14 cases (4 IgM, 1 IgG, 1 IgA). CD5 was expressed in 6/14 cases by flow cytometry. CD19 and 20 were uniformly positive, CD79b was variable and 12/14 cases tested did not express ROR1. Conclusion: This prospective study outlines the value of a targeted NGS panel in enhancing the diagnosis of unclassifiable LI B-NHL thereby improving patient access to novel therapies and clinical trials. It highlights recurrent MYD88 mutations in a subset of patients that have splenomegaly as a frequent feature. 5 year clinical follow up data, currently being gathered in both the MYD88 mutated and overall cohort, will be valuable in further characterisation and risk stratification to inform management of these patients. Disclosures Furtado: Abbvie: Other: Conference Support. El-Sharkawi:Abbvie: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Innate: Consultancy. Iyengar:Abbvie: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Janssen: Honoraria; Beigene: Consultancy.

Tracing CLL-biased stereotyped immunoglobulin gene rearrangements in normal B cell subsets using a high-throughput immunogenetic approach

BackgroundB cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5+ and CD5− B cells from the spleen and peripheral blood (PB).MethodsFractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5+ and CD5− cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution.ResultsCBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5+ B (0.57%) cells compared to CD5− B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family.ConclusionsCBS-IG receptors appear to represent a part of the “public” BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.

Prognostic impact of prevalent chronic lymphocytic leukemia stereotyped subsets: analysis within prospective clinical trials of the German CLL Study Group (GCLLSG).

Almost one-third of all patients with chronic lymphocytic leukemia (CLL) express stereotyped B-cell receptor immunoglobulins (BcR IG) and can be assigned to distinct subsets, each with a particular BcR IG. The largest stereotyped subsets are #1, #2, #4 and #8, associated with specific clinico-biological characteristics and outcomes in retrospective studies. We assessed the associations and prognostic value of these BcR IG in prospective multicenter clinical trials reflective of two different clinical situations: (i) early-stage patients ('watch and wait' arm of the CLL1 trial) (n=592); (ii) patients in need of treatment, enrolled in three phase III trials (CLL8, CLL10, CLL11), treated with different chemo-immunotherapies (n=1,861). Subset #1 was associated with del(11q), higher CLL International Prognostic Index (CLL-IPI) scores and similar clinical course to CLL with unmutated immunoglobulin heavy variable (IGHV) genes (U-CLL) in both early and advanced stage groups. IGHV-mutated (M-CLL) subset #2 cases had shorter time-to-first-treatment (TTFT) versus other M-CLL cases in the early-stage cohort (hazard ratio [HR]: 4.2, confidence interval [CI]: 2-8.6, P<0.001), and shorter time-to-next-treatment (TTNT) in the advancedstage cohort (HR: 2, CI: 1.2-3.3, P=0.005). M-CLL subset #4 was associated with lower CLL-IPI scores and younger age at diagnosis; in both cohorts, these patients showed a trend towards better outcomes versus other MCLL. U-CLL subset #8 was associated with trisomy 12. Overall, this study shows that major stereotyped subsets have distinctive characteristics. For the first time in prospective multicenter clinical trials, subset #2 appeared as an independent prognostic factor for earlier TTFT and TTNT and should be proposed for risk stratification of patients. (Trials registered at clinical trials. gov identifiers: NCT00262782, NCT00281918, NCT2000769522, and NCT01010061).

Higher Order Restrictions of the Immunoglobulin Repertoire in CLL: The Illustrative Case of Stereotyped Subsets #2 and #169

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.

High Frequency Mutations of MYD88 and KMT2D in Chinese Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) is one of the most common forms of adult leukemia in the Western population but rare in Chinese descent. As Chinese CLL patients have distinct clinical features, higher frequency of mutated IGHV as well as unique BCR stereotype, we hypothesize that genetic alterations may contribute to the disease phenotype differences. We profiled the known recurrent mutated genes associated with Western CLLs in 338 Chinese CLL samples, with the goal to define the somatic mutation pattern in the Chinese CLL population. Methods: We first assessed 96 CLL mutated genes in a discovery cohort that includes 126 CLL samples using Illumina MiSeq platform. We then validated 20 recurrent mutated genes in a validation cohort that comprises of 212 CLL samples with Ion Torrent platform. The presence of mutation in DNA and mRNA were further validated by Sanger sequence. Results: A total of 303 untreated and 35 relapsed samples were investigated. The average sequencing depth was 1957X (range: 112 to 4254). In the discovery cohort, we identified recurrently mutated genes in TP53 (17.5%), SF3B1 (14.3%), ATM (13.7%), KMT2D (12.7%), MYD88 (10.3%), NOTCH1 (8.7%), FBXW7 (8.7%), BIRC3 (7.9%), POT1 (4.8%), KRAS (4.0%). This was further confirmed in the validation cohort. In comparison to the frequency of these mutations in the western studies, we observed an unexpected significantly higher frequency of mutations in MYD88 and KMT2D (Chi-squared test, p&lt;0.01). Mutations of KMT2D occurred with a similar frequency in treatment-naïve (8%) and relapsed samples (9%). 19/30 (63%) mutations were loss-of-function as they are predicted to generate truncated proteins due to lacking part or all of the C-terminal domains (Figure 1A). We observed a significant co-occurrence between KMT2D mutations and trisomy 12 (Chi-squared test, p&lt;0.01, Figure 1E), suggesting there could be intrinsic linkage between these lesions. Further integration with IGHV mutation status revealed that CLLs with KMT2D mutations were characterized by an overrepresentation of VH4-34 and VH4-39 (p&lt;0.01, p=0.03, respectively) and enriched in stereotyped subset #8 (4/12, 33.3%) (Figure 1B). Consistent with previous reports that VH4-34 and VH4-39 are commonly used in Asians CLLs, we confirmed the high frequency of subset #8 in Chinese patients [1], indicating KMT2D as a uniquely mutated gene in Chinese CLLs. MYD88 mutations were found in 38/303 (13%) untreated CLLs. 17/38 (45%) of the mutations were at the L265P site (Figure 1C). Additional hotspot mutations were also detected (V217F 29%; S219C 13%). These mutations are associated with a significant male predominance (Chi-squared test, p&lt;0.01), increased monoclonal gammopathy (15%, p=0.01), and a higher rate of Hepatitis B virus (HBV) infection (HBV core antibody positivity 56%, p=0.02). Most of these mutations (87%) were clonal and IGHV mutated (97%). It is worth to note that the usage of VH3-7 and VH4-34 were more dominant in patients with MYD88 V217F/S219C (6/16, 38%) and with L265P (6/17, 35%), respectively. Mutations of MYD88 are mutually exclusive with TP53 mutation and del(17p) (Chi-squared test, p&lt;0.01). Moreover, we found a superior time to first treatment in MYD88-mutated patients compared with wide-type patients (Log-rank test, p=0.03). To further understand the linkage between mutations and clinical outcome, we divided 303 treatment-naive patients into two groups: Non-progressor (NPRO, 39%) and Progressor (PRO, 61%). NPRO and PRO were classified as patients who developed symptoms that need treatment either more than or less than three years after sampling. Epigenetic modifiers pathway mutations (KMT2D, CREBBP, EP300, TET2, ARID1A and IKZF1) were more frequently in PRO group (17% vs. 8%, p=0.02). Inflammatory pathway mutations (MYD88, BIRC3, CARD11 and TRAF3) was more involved in NPRO group, compared to PRO or Relapsed samples (21%, 15% and 0%, p&lt;0.01). Conversely, DNA damage pathway mutations (TP53, ATM and POT1) was more frequently found in Relapsed and PRO groups compared to NPRO group (49%, 30% and 7%, p&lt;0.01). Conclusion: In summary, we observed high frequency mutations in KMT2D and MYD88 that correlated with specific IGHV repertoire in Chinese CLL population. This suggests that pathogenesis of CLL between the Chinese and Western population may be different and potentially have therapeutic value. [1] Marinelli M, et al. Oncotarget. 2016 Apr 12;7(15):20520-31. Figure 1 Disclosures No relevant conflicts of interest to declare.

Changes in N-Glycosylation Induced By Somatic Hypermutation Modulate the Antigen Reactivity of the Immunoglobulin Receptors in CLL Stereotyped Subset #201

The IGHV4-34 gene is intrinsically autoreactive due to carrying a germline(GL)-encoded (super)antigenic motif binding various self (and exogenous) antigens, while it is one of the few IGHV genes that contain a GL-encoded N-glycosylation (N-glyc) site. IGHV4-34 is overrepresented in chronic lymphocytic leukemia (CLL), particularly in cases expressing B cell receptor immunoglobulin (BcR IG) with a significant load of somatic hypermutation (SHM; 'mutated' CLL, M-CLL). Moreover, a large fraction of IGHV4-34 M-CLL cases are clustered in different stereotyped subsets, of which the best studied is subset #4, the largest within M-CLL, defined by the expression of IgG-switched IGHV4-34/IGKV2-30 BcR IG with a distinctive SHM imprint. Considerably smaller than subset #4 is subset #201, defined by the expression of IGHV4-34/IGLV1-44 BcR IG of the IgMD isotype. Subset #201 is noteworthy owing to recurrent replacement SHMs that frequently lead to the creation of novel N- glyc motifs within the VH domain. This may be functionally relevant, considering that N-linked glycosylation is a widespread post-translational modification that is largely SHM-induced during antigen-specific immune responses and can modulate antibody (Ab) affinity towards antigen. That said, nothing is yet known about the antigen reactivity of subset #201 BcR IG and whether/how it could be affected through SHM-induced changes of N-linked glycosylation. In order to obtain insight into this issue, 4 subset #201 clonotypic IGs were expressed as recombinant monoclonal Abs (mAbs) of the mu isotype in HEK293 human cells, in either the authentic SHM state ('wildtype', WT-mAbs) or after reverting specific SHMs that altered N-glyc sites (R-mAbs) by site-directed mutagenesis. Since not all N-glyc motifs are eventually glycosylated, we used the NetNglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) for the prediction of N-glycan occupancy. Binding to MEC1 B CLL, Jurkat T and HEK293 cells was assessed by flow cytometry. Reactivity against nuclear Hep-2 cell extract, nDNA, actin, myosin, thyroglobulin (TG), β-amyloid, carbonic anhydrase, F(ab')2 and the non-self hapten trinitrophenyl was tested by ELISA. Non-subset #201 M-CLL mAbs (n=14, including 3 subset #4 mAbs), were used as controls. None of the subset #201 WT-mAbs displayed reactivity in any of the ELISAs. However, unlike most CLL mAbs, all subset #201 WT-mAbs bound to live MEC1 cells, while also exhibiting reactivity to HEK293 cells that was significantly higher when compared to non-subset #201 M-CLL (p=0.0095) or subset #4 (p=0.05); additionally, 1/4 subset #201 mAb displayed weak binding to Jurkat T cells. Three of 4 subset #201 mAbs bore a novel N-glyc site introduced by SHM in codons VL CDR1 36-38 of the clonotypic lambda light chains. Reversion to the GL in one such mAb resulted in enhanced binding to all 3 cell lines [fold change (FC) of binding of the R- vs WT-mAb to MEC1, Jurkat and HEK293: 1.3, 7.9 and 3.3, respectively) and in strong anti-TG activity. The GL-encoded N-glyc site in VH CDR2 57-59, that has been reported to be mostly unoccupied, was targeted by SHM in 2/4 subset #201 mAbs: reversion to GL decreased binding to both MEC1 and HEK293 cells (FC: -8 and -1.4 respectively). Finally, in 2/4 cases, SHM at codons VH FR3 67-68 inserted an N-glyc site that, however, is not predicted to acquire N-glycans. Reversion to GL enhanced the binding of one of these mAbs to MEC1 and HEK293 cells (FC: 2.1 and 5.6, respectively). The same mAb bore an additional predicted N-glyc site introduced by SHM at VH FR3 90-92; reversion of this change to GL augmented binding to both MEC1 and HEK293 cells (FC: 4.1 and 9.7, respectively). Double reversion of both aforementioned SHMs conferred further increased binding than any of the single reversions, implying a synergistic effect. Acquisition of novel N-glyc sites is not an intrinsic characteristic of either M-CLL in general or IGHV4-34 M-CLL in particular and its high incidence in subset #201 implies a selective process likely due to distinct (auto)antigenic pressure. Indeed, subset #201 mAbs exhibit an antigen reactivity profile that differs from that of typical polyreactive mAbs, including natural autoantibodies and other CLL mAbs, binding selectively to viable lymphoblastoid cell line cells and human HEK293 epithelial cells. These results further emphasize the importance of SHM in shaping the distinct (auto)antigenic recognition profile of CLL mAbs. Disclosures Chatzidimitriou: Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Targeted Next Generation Sequencing Reveals Molecular Heterogeneity in Non-CLL Clonal B-Cell Lymphocytosis

Background. Non-chronic lymphocytic leukemia (non-CLL) clonal B-cell lymphocytosis (CBL) encompasses a heterogeneous group of hematologic disorders that are still poorly understood. The main purpose of this study was to explore the genomic landscape of non-CLL CBL, correlating the results with clinical, histological and immunogenetic features. Methods. We retrospectively analysed the disease characteristics in a highly selected series of 28 patients, who had a clonal lymphocytosis in peripheral blood (PB) and clonal lymphoid populations in the bone marrow (BM), without evidence of lymphadenopathy, splenomegaly, peripheral cytopenia or constitutional symptoms.In all patients the BM evaluation, comprising flow cytometry, histology and immunohistochemistry, was not able to classify the lymphoproliferative disorder in any of the WHO-defined entities. A diagnosis of CLL or a leukemic phase of mantle cell or follicular lymphoma was ruled out in each subject. Targeted next generation sequencing (NGS) was performed on DNA extracted from CD19+ BM or PB cells using a core panel of 138 genes frequently reported in mature B cell neoplasms.On the basis of the information retrieved from the KEGG and REACTOME databases, each mutated gene was assigned to a specific lymphoma-enriched pathway. Results: Clinical, flow cytometric and histological data are presented in Table 1.The median BM infiltration was 30% (range: 5-80%); the pattern of infiltration was interstitial or mixed (nodular and interstitial) in the majority of the cases; sinusoidal localization was noticed in 54% of cases. There was a borderline correlation between the extent of BM infiltration and the absolute number of PB clonal B cells count (Spearman's Rho: 0.38, p=0.05), that ranged from 851/mmc to 14508/mmc (median: 3604/mmc). With a median follow-up of 35.2 months (range: 6-125), only 1 patient developed disease progression to a B-cell lymphoma not otherwise specified (5-yrs CI of progression: 11.9%; 95%CI: 0.6%-40.7%). The sequence analysis of 138 genes revealed a total number of 74 somatic mutations in 51 genes. All the 28 cases displayed at least 1 mutation, with the number of mutations ranging from 1 to 7 (median 2). The most frequently mutated genes were MYD88 (14%), PDE4DIP (14%), BIRC3 (11%), CCND3 (11%), NOTCH1 (11%) and TNFAIP3 (11%) (fig.1).Notably, 3 out of 4 MYD88 variants were different from the classical p.L265P (p.M232T, p.V217F and p.L204F not previously described). Data on IGHV were available for 22 patients (79%) (Fig.2): VH gene repertoire was similar to that reported in non-CLL malignancies and no distinct stereotyped subset was found. Truly unmutated IGHV genes with 100% of germline identity were present in 3 cases (11%). The analysis of disease features according to mutated genes showed that MCwas more frequent in patients harbouring mutations in MYD88 (p=0.08) or TNFAIP3 (p=0.02); these patients had also a higher number of mast cells on BM histology than the wild type ones (p=0.02 and p=0.05, respectively). NOTCH1 mutated patients showed an absolute number of B-cell peripheral clonal lymphocytes higher than wild type cases (p=0.037). The most frequently involved pathways were NF-kB signalling (32%) and cell communication pathways (32%), followed by cell cycle (29%), chromatin organization (25%), transcriptional misregulation (21%), Toll-like receptor (TLR) signalling (18%) and NOTCH signalling pathways (14%) (Fig. 3).Patients with mutated chromatin organization pathway more often showed normal LDH levels compared with wild type cases (100% vs 40% p=0.01). Conclusions. We characterised non-CLL CBL, integrating BM histological features and IGHV data with an extended NGS panel. The mutational landscape was extremely heterogeneous, pointing out a wide spectrum of somatic mutations. Most of them have been already described in Waldenström's Macroglobulinemia and splenic B-cell lymphomas; of note, we found a new variant of MYD88 associated with an IgM MC and recurrent mutations of PDE4DIP, which have not been previously reported in indolent B-cell malignancies. Disclosures Varettoni: Janssen: Consultancy; Roche: Consultancy; ABBVIE: Other: travel expenses; Gilead: Other: travel expenses. Arcaini:Bayer, Celgene, Gilead Sciences, Roche, Sandoz, Janssen-Cilag, VERASTEM: Consultancy; Celgene: Speakers Bureau; Celgene, Roche, Janssen-Cilag, Gilead: Other: Travel expenses; Gilead Sciences: Research Funding.

Distinct immunoglobulin heavy chain variable region gene repertoire and lower frequency of del(11q) in Taiwanese patients with chronic lymphocytic leukaemia.

SummaryChronic lymphocytic leukaemia (CLL) is the most common leukaemia in Western countries but very rare in Asia. Peripheral blood or bone marrow mononuclear cells obtained at initial diagnosis from 194 patients with CLL were analysed to determine the ethnic difference in genetic abnormalities. Mutated IGHV was detected in 71·2% of Taiwanese CLL and IGHV3‐23 was the most frequently used gene. Stereotyped BCR was present in 18·3% with subset 8 being the most frequent. All cases with subset 8 belonged to IGHV 4‐39 and were exclusively associated with un‐mutated IGHV and poor outcome. Mutation frequencies of SF3B1 (9·7%), NOTCH1 (8·6%), BIRC3 (1·1%), ATM (16·9%) or TP53 (8·1%), and frequencies of cytogenetic abnormalities including trisomy 12 (18·6%), del(17p) (10·4%), del(13q) (43·7%) and IGH translocation (10·1%) were comparable to those reported from Western countries, except del(11q) (6·9%) which was lower in our patients. Patients with un‐mutated IGHV, subset 8, disrupted TP53, trisomy 12, and SF3B1 mutations had a worse outcome compared to patients without these mutations. In conclusion, IGHV3‐23 usage, stereotyped subset 8 and lower frequency of del(11q) show an ethnicity‐dependent association in Taiwanese CLL patients.

PF365 PROGNOSTIC IMPLICATIONS OF IGHV GENE REARRANGEMENTS WITH “BORDERLINE” PERCENTAGE OF MUTATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

Background:The somatic hypermutation (SHM) of immunoglobulin heavy chain variable (IGHV) region genes classifies chronic lymphocytic leukemia (CLL) patients into two subgroups: unmutated CLL (UM‐CLL, ≥98%) associated with an adverse prognosis and mutated CLL (M‐CLL, &lt;98%) showing a good outcome. In cases with a % of IGHV identity close to the 98% cut‐off, defined as “borderline” (BL‐CLL), the prognosis remains controversial.Aims:To investigate the clinico‐biologic features and prognosis of BL‐IGHV CLL in view of their undefined outcome.Methods:We retrospectively analyzed 811 untreated CLL patients, including 327 at diagnosis, sequenced at our Institution. BL‐CLL were defined as cases with a 97–97.9% IGHV identity, according to the 2017 European Research Initiative on CLL (ERIC) recommendations. IGHV were amplified using family‐specific VH primers (framework region 1 [FR1] or leader primers) and sequenced using the 3500 Series Genetic Analyzer. Following the ERIC recommendations, we updated the IGHV analysis with two different approaches in order to accurately determine the SHM rate: 1) re‐sequencing of 82 old FR1 cases with leader primers; 2) realignment of all cases to the updated IMGT/V‐QUEST tool. From the re‐analysis with leader primers of 82 old FR1 sequenced cases, none shifted the UM/M IGHV category, while 6 (7.3%) were reclassified as follows: 4 M‐CLL as BL‐CLL and 2 BL‐CLL as UM‐CLL. No case shifted across the three categories after the realignment to the updated IMGT/V‐QUEST tool. Major stereotyped subset analysis was determined according to the ARResT/AssignSubsets tool (bat.infspire.org/arrest/ericll.org/pages/services/tool). Time‐to‐first treatment (TFT) was evaluated as a clinical endpoint. Two further independent cohorts of 308 and 465 newly diagnosed CLL patients were used to validate the data.Results:In the 811 untreated patients, 418 (51.5%) UM‐CLL, 39 (5%) BL‐CLL and 354 (43.5%) M‐CLL were identified, with a higher frequency of UM‐CLL due to the enrichment of cases enrolled in first line clinical trials. Sixteen cases (2%) belonged to stereotyped subset #2. BL‐CLL showed a similar proportion of TP53 deletion and mutation compared to UM‐CLL, but a significant lower frequency of other unfavorable prognostic factors (stage, CD38/ZAP70 expression) (Table 1). Moreover, BL‐CLL were enriched in subset #2 (5/39, 13%), in agreement with the higher incidence of subset #2 among these cases. Applying the conventional 98% IGHV cut‐off, TFT was significantly shorter in UM‐CLL than M‐CLL (p &lt; 0.0001), as well as in subset #2 CLL vs M‐CLL (p = 0.017), as expected. When analyzing the 97–97.9% IGHV subgroup, BL‐CLL showed a significantly longer TFT than UM‐CLL (p &lt; 0.0001), similar to that of M‐CLL (Fig. 1a). The TFT of BL‐CLL cases remained comparable to that of M‐CLL, also when the analysis was restricted to the 327 CLL patients at diagnosis (80% Binet stage A) (125 UM‐CLL, 19 BL‐CLL and 183 M‐CLL) (Fig. 1b), as well as excluding subset #2 cases. These findings were validated in two independent cohorts of CLL at diagnosis (105 UM‐CLL, 16 BL‐CLL, 187 M‐CLL; TFT BL vs UM‐CLL: p &lt; 0.0001 and 143 UM‐CLL, 13 BL‐CLL, 309 M‐CLL; p = 0.024), that included 719 Binet stage A CLL.Summary/Conclusion:Our data suggest that the prognosis of BL‐CLL is better than that of UM‐CLL and similar to that of M‐CLL, thus supporting the 98% cut‐off for clinical purposes. The issue of BL‐CLL remains open to further investigations.image

PS1131 HIGH‐THROUGHPUT B‐CELL IMMUNOPROFILING AT DIAGNOSIS AND RELAPSE OFFERS FURTHER EVIDENCE OF FUNCTIONAL SELECTION THROUGHOUT THE NATURAL HISTORY OF CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Chronic lymphocytic leukemia (CLL) is divided into two broad prognostic categories, namely mutated (M) and unmutated (U) CLL, according to the somatic hypermutation (SHM) status of the clonotypic heavy chain immunoglobulin (IGHV) gene. This is perceived to remain stable over time, as evidenced by low‐throughput studies, which however precluded investigation of subclonal architecture and evolution overtime.Aims:Here, we sought to comprehensively assess the B cell receptor (BcR) IG gene repertoire at CLL diagnosis and 1st relapse after chemoimmunotherapy (FCR) by next‐generation sequencing (NGS).Methods:We studied 5 patients, of whom 1 each expressed BcR IG typical of stereotyped subset #1 (U‐CLL), subset #4 (M‐CLL) or subset #6 (U‐CLL), whereas the remaining 2 concerned non‐stereotyped U‐CLL. Genomic DNA was amplified by multiplex PCR and products were subjected to paired‐end NGS. Sequence data were processed by a validated bioinformatics pipeline performing strict quality filtering. Rearrangements with identical IGHV gene and CDR3 amino acid (aa) sequence were defined as clonotypes. The dominant clonotype was defined as major, and clonotypes with the same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3 were considered as its satellite subclones.Results:In total, we analyzed 1,682,728 filtered‐in, productive IGHV‐IGHD‐IGHJ gene rearrangements (median 195,460 rearrangements/sample). In all cases, the major clonotype (IGHV and CDR3 aa sequence) was identical to that determined by Sanger sequencing and remained the same at diagnosis and relapse. However, it consisted of multiple distinct nucleotide (nt) sequences; while most nt sequences displayed the same SHM load as with Sanger (median 73.9% of all nt sequences of the major clonotype), there were other nt sequences with higher or lower SHM load. In all cases, nt sequences with discordant SHM status were practically negligible (median frequency 0.01%). Notably, the relative frequency of the dominant nt sequence increased at relapse for all cases (median increase by 3.7%, p &lt; 0.01). A particular note for subset #4, which by definition displays high SHM, was the emergence of major clonotype nt sequences with lower or even absent SHM at relapse. The median frequency of the major clonotype at diagnosis was 92.5% (range 86.1–93.3%) and remained stable at relapse (92.6%, range 89.9–93.8%). In all cases, the major clonotype came along with numerous satellite subclones (same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3) at both diagnosis and relapse (median n = 685 vs n = 604 respectively, p&gt;0.05). When considering both the major clonotype and its satellite subclones, the respective cumulative frequency corresponded to nearly the entire B cell repertoire/sample (median 99.6%, range 97.6–99.8%). Subset #4 satellite sublones displayed recurrent SHM patterns within the CDR3 at relapse, which actually diverged from the major clonotype and converged towards the consensus CDR3 sequence for this subset as defined by 176 different subset #4 patients.Summary/Conclusion:Overall, our study provides insight into the evolving architecture of the BcR IG repertoire of relapsing CLL, revealing that, similar to genomic subclones, there is a universe of BcR IG subclones as well. While the possibility of technical error cannot be excluded, the identification of recurrent SHM patterns and trends at relapse strongly points towards functional selection.

Stereotyped B Cell Receptor Immunoglobulins in B Cell Lymphomas.

Comprehensive analysis of the clonotypic B cell receptor immunoglobulin (BcR IG) gene rearrangement sequences in patients with mature B cell neoplasms has led to the identification of significant repertoire restrictions, culminating in the discovery of subsets of patients expressing highly similar, stereotyped BcR IG. This finding strongly supports selection by common epitopes or classes of structurally similar epitopes in the ontogeny of these tumors. BcR IG stereotypy was initially described in chronic lymphocytic leukemia (CLL), where the stereotyped fraction of the disease accounts for a remarkable one-third of patients. However, subsequent studies showed that stereotyped BcR IG are also present in other neoplasms of mature B cells, including mantle cell lymphoma (MCL) and splenic marginal zone lymphoma (SMZL). Subsequent cross-entity comparisons led to the conclusion that stereotyped IG are mostly "disease-specific," implicating distinct immunopathogenetic processes. Interestingly, mounting evidence suggests that a molecular subclassification of lymphomas based on BcR IG stereotypy is biologically and clinically relevant. Indeed, particularly in CLL, patients assigned to the same subset due to expressing a particular stereotyped BcR IG display remarkably consistent biological background and clinical course, at least for major and well-studied subsets. Thus, the robust assignment to stereotyped subsets may assist in the identification of mechanisms underlying disease onset and progression, while also refining risk stratification. In this book chapter, we provide an overview of the recent BcR IG stereotypy studies in mature B cell malignancies and outline previous and current methodological approaches used for the identification of stereotyped IG.

Intraclonal Diversification Occurs in Chronic Lymphocytic Leukemia Expressing B Cell Receptors Belonging to the IGHV4 Gene Family

Background. The pivotal role of the Immunoglobulin (Ig) receptor and antigenic stimulation have been proven to be landmarks for the understanding of the ontogeny and evolution of chronic lymphocytic leukemia (CLL). In addition, the mutational status of the Immunoglobulin Heavy-Chain Variable region gene (IGHV) was confirmed to be a reliable prognostic factor, supporting an antigen-driven model of CLL development. To clarify aspects regarding an antigenic involvement in CLL evolution, studies focusing on intraclonal diversification (ID) of Ig genes have provided relevant information, although mainly conducted in a pre-Next Generation Sequencing (NGS) era.Aim. To apply a NGS approach to investigate ID in CLL.Methods. IGHV genes from 530 CLL patients with Royal Masden Hospital score 4-5 (Fig. 1A) was sequenced using NGS (Lymphotrack). The bio-informatic pipeline was based on the pRESTO/ChangeO packages. Specific pathological clones were selected based on the presence of same IGHV, junction genes and with similar HCDR3 sequence according to Hamming's distance. Through the R-Alakazam package, we generated rarefaction curves to evaluate the clonal diversity inside the pathological clone (Fig. 1B). Focusing on the Simpson index (represented by the Hill number of order q=2), which gives more weight to larger clones minimizing the smaller ones (Fig. 1B), we selected a Diversity Score (DS) of 4 for the definition of cases without ID (clonal; DS <4) and cases with ID (intraclonal; DS ≥4) (Fig. 1B).Results. Using the reported threshold we identified 469 (88.5%) clonal cases, expressing a single clone (Fig. 1C), and 61 (11.5%) cases with ID (median DS 9.2, range 4.4-66.0) characterized by the presence of two or multiple pathological clones expressing the same IGHV gene and HCDR3 (Fig. 1C). Notably, cases with ID expressed both a mutated (M) (39/61, 63.9%) and an unmutated (UM) (22/61, 36.1%; p=0.066) IGHV gene configuration (Fig. 1C). Of note, we observed a significant skewing toward the usage of VH4-family genes when comparing cases with ID (38/61, 62.3%) vs. cases without ID (78/469, 16.6%; p<0.0001, Fig 1D). Moreover, the IGHV4-39 and IGHV4-34 genes were the most used genes in the context of cases with ID (Fig. 1E), although none of them belonging to known stereotyped subsets. By focusing on VH4-family only cases, we observed that cases with ID and UM IGHV genes displayed higher mutation frequencies in WA/TW motifs, a mutational signature which suggests an involvement of both Activation-Induced (Cytidine) Deaminase (AID) and error-prone polymerase eta (Fig. 1F), a pattern not observed in its counterpart with UM IGHV genes but without ID (Fig. 1F). Conversely, in cases with ID and M IGHV genes, mutations preferentially clustered in AID hotspots (WRC/GYW motifs), suggesting a direct role of AID and the Base Excision Repair machinery in the mutational overload (Fig. 1F). Consistently, M IGHV cases with ID expressed significantly higher AID mRNA levels than M IGHV cases without ID (p=0.0024; Fig. 1G). These expression levels were overall comparable with those found in UM IGHV cases, irrespective to the evidence of ID (Fig. 1G), which however were not associated with an increased number of mutations in AID-specific hotspots (Fig. 1F).Conclusions. By taking advantage of a new method for ID assessment in CLL, we demonstrated that ID prevalently affects VH4-family cases which display different mutational patterns dependent to the IGHV gene status. This data are in keeping with previous reports indicating the IGHV4 genes as particularly prone to generate immunoglobulin subjected to continuous/persistent stimulation by external/auto-antigens, hence particularly prone to generate features of ID. Further experiments in selected cases with ID through a non-random barcode strategy are needed. DisclosuresZaja:Sandoz: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Abbvie: Honoraria.

Remarkable Functional Constraints on the Antigen Receptors of CLL Stereotyped Subset #2: High-Throughput Immunogenetic Evidence

Subset #2 is the largest subset carrying stereotyped B cell receptor immunoglobulin (BcR IG) in chronic lymphocytic leukemia (CLL). This particular BcR IG is composed of heavy (HC) and light (LC) chains encoded by the IGHV3-21 and the lambda IGLV3-21 gene, respectively. The clonotypic IGHV3-21 genes display a variable load of somatic hypermutation (SHM), being mostly classified as mutated (M-CLL) but also including unmutated (U-CLL) cases. Subset #2 cases, independently of the SHM status, have a particularly dismal clinical outcome similar to that of patients with TP53 aberrations, although lacking such aberrations. Subset #2 BcR IG display a series of distinctive features, including conservation at certain VH and VL CDR3 positions and recurrent SHMs; as well as a capacity for self-association leading to cell autonomous signaling that is critically dependent on a substitution of Arginine (R) for Glycine (G) introduced by SHM at the lambda VL-CL linker region. These features implicate antigen selection in CLL subset #2 ontogeny. However, the available molecular evidence derives from low throughput immunogenetic analysis, precluding comprehensive assessment of antigenic impact on (sub)clonal composition. Here, we sought to overcome this limitation by performing next-generation sequencing (NGS) of HC and LC gene rearrangements of 20 subset #2 patients. RT-PCR products amplified by the IGHV3-21/IGHJ6 and IGLV3-21/IGLC primer pairs, respectively, were subjected to NGS on the MiSeq Illumina Platform. NGS data was analyzed by a validated bioinformatics pipeline. Rearrangements with identical CDR3 amino acid (aa) sequences were defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. Starting with HCs, we obtained 3,340,508 (mean: 291,751, range: 101,231-186,055) productive reads. On average, each analyzed sample carried 92 distinct clonotypes (range: 71-152), with the dominant clonotype having a mean frequency of 96% (range: 67-99%): in all cases the dominant clonotype was identical to that determined by Sanger sequencing. The dominant clonotype displayed considerable intraclonal heterogeneity with a mean of 5,082 subclones/sample (range: 2,946-11,041). Turning to LCs, we obtained 5,094,045 (mean: 231,547, range: 38,036-507,949) productive reads. LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean 222, range: 156-306). The dominant clonotype had a mean frequency of 96% (range: 74-98%); similar to HCs, it was identical to that determined by Sanger sequencing. Intraclonal heterogeneity was observed in the LCs as well, with a mean of 7,382 subclones/sample (range: 1,946-11,866), hence more pronounced vs their partner HCs. Viewing the entire subset #2 VH or VL CDR3 dataset (i.e. the CDR3 aa sequences from all clonotypes of all cases) as a single entity branching through diversification enabled the identification of 2 distinct VH CDR3 sequences present at varying frequencies in 16 and 13 cases, respectively; and, 3 distinct VL CDR3 sequences present at varying frequencies in all 20 cases: these results allude to important constraints on the composition of the antigen binding site. Focusing on SHM, the following notable observations could be made. (i) The G-to-R substitution at the VL-CL linker was a clonal event in all cases with R being degenerately encoded by different nucleotide sequences; altogether, these findings underscore the seminal role of this recurrent SHM, likely due to mediating self-association. (ii) A recurrent 3-nucleotide deletion was detected in the VH CDR2 of all cases, strongly supporting functional pressure. This change, previously identified by Sanger sequencing as a recurrent SHM in subset #2 (albeit at a frequency of only 25%), was clonal in 4 cases and subclonal in the remainder, where it was present in an average of 105 subclones/sample (range: 1-369). (iii) Certain positions in both the VH and VL domain bore the same aa substitution, mostly at subclonal level: the prime example concerned the G for Serine (S) substitution within the VL CDR3, detected in all samples at a mean frequency of 44.2% (range: 6.3-87%). In conclusion, we provide compelling immunogenetic evidence for functional pressure in the ontogeny of CLL subset #2. On this evidence, subset #2 emerges as perhaps the most striking example of antigen-driven leukemogenesis reported thus far. DisclosuresGemenetzi:Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Hadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.

IGHV Gene Replacement: A Potential Mechanism for Establishing Stereotypy in Certain Cases of Chronic Lymphocytic Leukemia

Immunoglobulin heavy chain variable gene (IGHV) replacement or “VH replacement” (VHR) modifies a rearranged IGHV-D-J sequence by replacing the original IGHV gene with another. This process leaves a detectible “footprint” at the IGHV-D junction of the existing sequence. Roughly 33% of chronic lymphocytic leukemia (CLL) cases exhibit stereotyped B cell receptors (BCRs) often characterized by signature VH CDR3 amino acids. Various mechanisms have been put forth to account for stereotypy in CLL. An overarching hypothesis is that the stereotyped BCRs are antigen driven. Within this concept, a variety of mechanisms could lead to the signatures including somatic mutations and addition/deletion of nucleotides at junctional regions. Here we explore the possibility that VHR provides another mechanism to account for some of the stereotyped rearrangements and some of their signature VH CDR3 amino acid residues in CLL.We examined IG sequences of 26,642 CLL cases and ~16 million healthy controls (HC) to find relic footprints as indicators of VHR. This was done using the VHRFA program developed by Lin Huang et al (PLoS ONE, 2013), as well as our own program which duplicates the VHRFA results but is better able to process large numbers of sequences.The frequency of VHR was similar in CLL and HC (11.6 and 11.9%, respectively). Focusing solely on CLL sequences to define a relationship between VHR and stereotypy, we found highly significant differences in VHR frequencies between stereotyped (n=8,568) and non-stereotyped cases (n=18,074), with stereotyped cases exhibiting VHR at a greatly reduced frequency (7.7% vs. 13.5%, respectively). When comparing VHR frequencies between stereotyped cases and non-stereotyped cases that used the same IGHV, we found that the number of subsets with low VHR exceeded those with elevated VHR ~2:1, accounting for the overall VHR in stereotyped cases being lower than non-stereotyped cases. Further restricting comparisons of stereotyped subsets to non-stereotyped cohorts by matching VH CDR3 length led to similar conclusions.Within stereotyped cases there was a wide distribution of VHR, ranging from 55.6% to 0.1%. Restricting VH CDR3 lengths to “short” (5 - ≤13), “medium” (13.1 - ≤20) and “long” (20.1 - ≤28), the corresponding VHR increased monotonically with length (1.1, 8.2, and 11.9% respectively). Notably, subsets showing elevated VH replacement included better prognosis subsets, #4, 77 and 201 (23.8, 22.1, and 28.6%, respectively). Among low VHR frequency subsets were those associated with worse prognosis, #1, 2, 5, 6, 8, 9 and 10 (VHR frequencies: 0.2, 0.1, 0.9, 2.3, 7.7, 9.0 %, respectively). This was most strikingly exhibited by subsets #1 and #2, both of which comprise patients with poor clinical courses. Each of these sets of sequences displayed virtually no examples of VHR (0.2 and 0.1%, respectively). This might be predicted because these two subsets have relatively short VH CDR3 lengths (subset #1: 13 aa; subset #2: 9 aa), based on the length association mentioned above.Detailed analyses of the presence of footprints and the position of these in the rearranged IGHV-D-J indicated that for some subsets, certain signature VH CDR3 amino acids could be the result of VHR. For example in subset #201, sequence analysis suggests that VHR is responsible for an arginine and for a glutamine in the 5‘ portion of the VH CDR3. Similarly, VHR may craft the characteristic glutamine on the 5‘ end of the subset #6 VH CDR3.Thus, our studies indicate that, as a whole, CLL IGHV-D-J sequences use VHR at a frequency comparable to that of normal B cells and significantly less than that of non-stereotyped rearrangements. However, certain stereotyped cases are dramatically enriched for evidence of VHR. Moreover among these cases, the footprints found in the VH CDR3s of stereotyped cases can be shown to directly code for signature amino acids in VH CDR3s. Finally, stereotyped cases with high levels of VHR tend to be those with better clinical courses, whereas those worse outcome stereotyped cases exhibit less evidence for this process. This latter finding is consistent with the concept that VHR is one of the molecular mechanisms used by developing B cells to edit BCRs having high affinity for autoantigens. Since many CLL BCRs are autoreactive, including those found to have high levels of VHR such as subset #4, this implies a fundamental defect in tolerance mechanisms in those normal B cells that eventually became leukemic. DisclosuresAgathangelidis:Gilead: Research Funding. Hadzidimitriou:Janssen: Honoraria, Research Funding; Gilead: Research Funding; Abbvie: Research Funding. Ghia:AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding. Stamatopoulos:Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

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.

ANALYSIS OF THE 3΄UTR REGION OF THE NOTCH1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS

Detection of mutations in the 3'UTR region of the NOTCH1was performed by direct sequencing in 87previously untreated CLL patients (from the total group of 237CLL patients) with unmutated immunoglobulin heavy-chain variable (UM IGHV) genes and without mutations in hotspot regions of TP53, SF3B1, and exon 34of NOTCH1genes. Mutations in the 3'UTR region of the NOTCH1were revealed in three of 87CLL patients (3.4%). Two cases with non-coding mutations were related to subset #1of stereotyped B-cell receptors, and one case belonged to stereotyped subset #28a. Analysis with inclusion of 30UM IGHV cases with previously detected c.7544_7545delCT mutations revealed that the frequency of UM IGHV genes of I phylogenetic clan (except IGHV1-69) was significantly increased, and the frequency of UM IGHV3and IGHV4genes, on the contrary, was reduced in NOTCH1-mutated cases comparing with NOTCH1-unmutated cases (p = 0.002) and the general group (p = 0.013). SNP rs3124591did not affect the risk of CLL and survival parameters of the patients. At the same time, differences were found in the frequency of IGHV gene usage and in the structure of HCDR3in carriers of individual genotypes. The frequency of NOTCH1mutations in 3'UTR region was low. Our findings confirmed current data on the association between the structure of the B-cell receptor and the appearance of NOTCH1mutations. Some features of HCDR3structure were identified in carriers of TT and CC genotypes of rs3124591.