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An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset # 2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset # 2. Within subset # 2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset # 2/IGHV3-21 was enriched for IGHV-unmutated cases (P =.002). Subset # 2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset # 2/IGHV3-21 (22 vs 60 months, P =.001). No such difference was observed between non-subset # 2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset # 2 emerges as uniformly aggressive, contrasting non-subset # 2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Somatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, “stereotyped” amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are underrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. The precise targeting and distinctive features of somatic hypermutation (SHM) in selected subgroups of CLL patients provide further evidence for selection by specific antigenic element(s).

In MM patients relapsing after MRD-negativity, the disease could reemerge from immature cells or from undetectable MRD. However, it remains unknown if immature cells have the same genetic background as MM plasma cells (PCs), as well as the amount of MRD that persists below the limit of detection (LOD) of next-generation techniques. To obtain further insight, we compared the biological landscape of MM PCs at diagnosis to that of CD34 progenitors, B cells and normal PCs isolated from patients with negative MRD by next-generation flow (NGF) after treatment. We performed whole-exome sequencing (WES, mean depth: 90x) with the 10XGenomics Exome Solution for low DNA-input as well as deep NGS of B-cell receptor immunoglobulin (BcR IG) gene rearrangements (mean, 69,975 sequences), in a total of 68 cell-samples isolated from the bone marrow (BM) of 7 MM patients with MRD-negativity by EuroFlow NGF after induction with VRD and auto-transplant (GEM2012MENOS65 trial). Patients with negative MRD were intentionally selected to avoid contamination with MM PCs during sorting of CD34 progenitors, B-cell precursors, mature B cells and normal PCs after induction and transplant. We investigated in these populations the presence of somatic mutations and clonotypic BcR Ig rearrangements detectable in MM PCs sorted at diagnosis, using peripheral blood T cells as germline control. We also performed WES in matched diagnostic MM PCs and MRD cells persisting after VRD induction in 14 cases as control. In another 6 patients with untreated MM, we performed single-cell RNA and BcR IG sequencing (scRNA/BcRIGseq) of total BM B cells and PCs (n=16,380) to investigate before treatment, if the clonotypic BcR IG sequence of MM PCs was detectable in other B cell stages defined by their molecular phenotype. We used multidimensional flow cytometry (MFC) to investigate the frequency of B cell clonality in BM samples from a larger series of 195 newly-diagnosed MM patients, prospectively enrolled in the GEM-CLARIDEX trial. Somatic mutations present in diagnostic MM PCs were detectable in the lymphopoiesis of 5/7 patients achieving MRD-negativity after treatment. In one case, out of 55 mutations present in diagnostic MM PCs, a single mutation in PCSK1N (VAF: 0.30) was detectable in normal PCs. In the other four patients, a total of 85 mutations were present in MM PCs and up to 10 (median VAF, 0.16) were found all the way from CD34 progenitors into B-cell precursors, mature B cells and normal PCs, but not in T cells. Of note, most mutations were reproducibly detected in each cell type after induction and after transplant. All somatic mutations shared by MM PCs and normal cells were non-recurrent, and genes recurrently mutated in MM (eg. ACTG1, ATM, DIS3, FAM46C, KRAS, LTB, MAX, TRAF3) were found in MM PCs but never in normal cells. Copy number alterations (CNA) were found only in MM PCs. By contrast, up to 513/827 (62%) mutations and 48/67 (72%) CNA were detectable in matched diagnostic MM PCs and persistent MRD cells, indicating that the few somatic variants present in normal cells were unlikely related to contaminating MRD below NGF's LOD. Accordingly, MM clonotypic BcR IG rearrangements were detectable in normal PCs (4/7patients) and in immature B cells (5/7 patients) but at much lower frequencies (mean of 0.02% in both). Of note, 9 additional clonotypes (mean 8.4%) were found in MM PCs of 5/7 patients (range, 1-3). scRNR/BcRIGseq unveiled that clonotypic cells were confined mostly but not entirely within PC clusters, and that in 1 patient another clonotype was detectable in mature B cells. Accordingly, using MFC we found in a larger series that 25/195 (13%) of newly-diagnosed MM patients display B-cell clonality (median of 0.7% BM clonal B cells, range 0.02%-6.3%). In conclusion, we show for the first time that MM patients bear somatic mutations in CD34 progenitors that specifically differentiate into the B cell lineage, likely before the disease onset. Because diagnostic, MRD (and relapse) MM PCs display great genetic similarity, these results suggest that undetectable MRD Disclosures Puig: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda, Amgen: Consultancy, Honoraria; The Binding Site: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Martinez-Lopez:BMS: Honoraria, Other: Advisory boards; Janssen: Honoraria, Other: Advisory boards and Non-Financial Support ; Amgen: Honoraria, Other: Non-Financial Support ; Celgene: Honoraria, Other: Advisory boards and Non-Financial Support ; Incyte: Honoraria, Other: Advisory boards; Novartis: Honoraria, Other: Advisory boards; VIVIA Biotech: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria. Lahuerta:Takeda, Amgen, Celgene and Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosinol Dachs:Janssen, Celgene, Amgen and Takeda: Honoraria. Bladé:Jansen, Celgene, Takeda, Amgen and Oncopeptides: Honoraria. Mateos:EDO: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Azacytidine (AZA), the mainstay of therapy in high risk Myelodysplastic syndromes (HR-MDS), affects CD4+ T-cell polarization and function, but the effect of these changes on tumor immunity is unclear. Signal transducer and activator of transcription (STAT) proteins are key regulators of differentiation and polarization of CD4+ T-cells in both health and cancer, but the STAT signaling architecture of CD4+ T-cell subsets in HR-MDS and its modulation by AZA are currently unknown. We applied single-cell phosphospecific flow cytometry in peripheral blood mononuclear cells from 67 HR-MDS patients at various time-points during AZA therapy. Unsupervised clustering of pretreatment STAT signaling profiles (SPs) of CD4+ T-cells revealed three signaling clusters (SCs), mainly differing in the potentiated responses of STAT3 to IL-6 stimulation (IL-6/STAT3 node). Compared to SC#1 and SC#3, patients in SC#2 displayed higher IL-6/STAT3 levels, higher levels of naïve (TN, p=0.05) and lower levels of PD1+ (p=0.04) and central memory ( TCM, p=0.04) CD4+ T-cells, and longer median overall survival (mOS, p=0.028, fig 1A). Moreover, comparisons of single signaling nodes revealed that the IL-6/STAT3 node correlated inversely with PD1+ (p=0.02) and IL-4+ (p=0.04) and positively with naïve CD4+ (p=0.04) and IFNγ+CD8+ T-cells (p=0.01). No other differences in clinicobiologic parameters, CD4+ and CD8+ T-cell subpopulations (FOXP3, IFNγ, IL-4, IL-17, Perforin and Helios) were noted among the 3 SCs and all other single nodes. To assess the effect of AZA on STAT signaling, we clustered the fold fold-change of pre- versus 6-month post-AZA SPs in CD4+ T-cells. Again the IL6/STAT3 node was the only differentiator among the clusters, and, by single node analysis, downregulation of IL6/STAT3 at 6th cycle (n=26) was associated with better response to AZA (p=0.02) and longer mOS (p=0.03), compared to upregulation of the same node (n=22); the latter also accompanied by an increase of IFNγ+CD8+ cells after AZA, (p=0.02, fig 1B). Further supporting a direct and beneficial modulation of the IL-6/STAT3 axis in CD4+ T-cells by AZA, the kinetics of IL-6/STAT3 during AZA therapy revealed a marked downregulation of the former node both at day15 (p=0.04) and cycle 6 after AZA (p=0.04) in responders (n=5), while no changes were observed in non-responders (n=7). We further compared the transcriptional profiles of isolated bone marrow CD4+ T-cells between responders (n=4) and non-responders to AZA (n=4) by RNA-seq, both prior and after AZA. No significant differences in pretreatment gene expression were identified. By contrast, 105 genes were differentially expressed at cycle 6 compared to pretreatment in responders (FDR To trace the molecular background associated with the differential regulation of the IL-6/STAT3 pathway we constructed mutational profiles by targeted DNA sequencing of 156 genes in blood mononuclear cells. No associations were found between pre or post-treatment IL-6/STAT3 node and mutational burden. By contrast, mutations in RNA splicing and STAG2 correlated with lower (p=0.02) and higher (p=0.017) pretreatment levels of IL6/STAT3, respectively. Notably, all 5 patients with NPM1/DNMT3A double mutation upregulated significantly IL6/STAT3 after AZA (p=0.03, fig 1D). Collectively, our results reveal for the first time that downregulation of the IL-6/STAT3 signaling axis in CD4+ T cells may represent an immune-mediated mechanism of action of AZA. However, the antileukemic activity of IL-6/STAT3LowCD4+ T-cells appears to be independent from modulation of common metrics of tumor immunity, as, paradoxically, the detrimental IL-6/STAT3 upregulation was linked with an expansion of antitumor T cell subsets and decrease of the immunosuppressive ones. The IL-6/STAT3 axis is notoriously pro-tumorigenic and pharmacologic inhibition of various individual modules of this pathway in cancer is under development. Our findings may serve as a guidepost for the ongoing investigation of IL-6/STAT3 axis inhibition as a therapeutic strategy to overcome azacytidine resistance in HR-MDS. Figure 1 Disclosures Vassilakopoulos: Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: 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; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; WinMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Pappa:Gilead: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding. Papaemmanuil:Celgene: Research Funding. Kotsianidis:Celgene: Research Funding.

Viral infections, mainly by cytomegalovirus (CMV), Epstein Barr virus (EBV) and polyomavirus type I (BKV), are major causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As effective immune responses against human viruses rely on an armamentarium of T-cell receptor (TR) repertoire capable of recognizing a broad range of antigenic peptides of those pathogens, reconstitution of antiviral immunity, either by spontaneous generation of endogenous virus-specific T cells (VSTs) or by adoptive immunotherapy with VSTs, plays a critical role to fight infections. We here evaluated the diversity and clonality of TR repertoire of functional tri-virus-specific T cell products generated from immunocompetent donors (n=10) and compared their TR gene repertoire to that of peripheral blood mononuclear cells (PBMCs) from patients who had undergone allo-HSCT (n=5). To generate tri-VSTs, PBMCs derived from 15-20ml of peripheral blood of normal donors, were exposed to EBV, CMV and BKV overlapping peptides and cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Specificity of donor-derived VSTs and patient-derived PBMCs was measured by IFN-γElispot. TR diversity was investigated by next-generation sequencing on a MiSeq Sequencer, after amplification of TR beta chain gene rearrangements by RT-PCR with the BIOMED-2 protocol. Raw NGS reads were filtered based on their length and quality and the filtered-in sequences were submitted to IMGT/HighVQUEST. Metadata analysis and clonotype computation were performed using a validated in-house bioinformatics platform. As clonotype we defined sequences carrying the same TRBV gene and identical CDR3 amino acid sequence. Tri-VSTs provided 947,298 productive TRBV-TRBD-TRBJ rearrangements and a polyclonal and highly diverse TR gene repertoire, consisting of a total of 169,502 unique clonotypes (average: 16,950/sample, range 4,057-45,602), 64,971 (38.3%) of which were expanded (corresponding to more than one sequence). In terms of clonality, the mean relative frequency of the major clonotype in all tri-VSTs was 12.6% (range 3.3-29.2%). Interestingly, among tri-VST cell lines, 637 clonotypes were shared (present in >2/10 samples), 80 were highly shared (present in >3/10 samples) while 7 were present in 6-8 different VST lines and largely expanded, accounting for up to 29.2% of all sequences. Importantly, there were 65 of 96 major VST clonotypes shared, thus suggesting that they were potentially associated with recognition of the targeted viruses. Given that 4/10 VSTs cell lines were not specific for CMV, while being EBV-and BKV-specific, dominant TRs in those 4 cell lines can potentially be associated with EBV- or BKV-activity. By searching a public database of TR clonotypes with known reactivity against EBV and/or CMV (ShugayM, Nucleic Acids Research, 2018), we found 8 shared EBV-specific and 4 shared CMV-specific clonotypes among our VSTs and the 499 public clonotypes. When we compared the produced VSTs with PBMCs from 3 allo-grafted patients with circulating CMV-, BKV- and EBV-specific T cells and previous viral reactivation, we detected 163 shared clonotypes. Likewise, we observed 21 and 23 shared clonotypes in similar frequencies, between VSTs and PBMCs from 2 patients with CMV- or BKV-specific T cell immunity. These data identify clones that potentially expand in vivo and protect patients from viral infections. Overall, our findings reveal high levels of TR clonality in cell lines enriched for T cells reactive against EBV and/or CMV and/or BKV and provide insights into the TR repertoire of ex vivo- or endogenously-generated VSTs. Our approach may help to identify optimal TRs for immunotherapy as well as TRs which can be used as a tool for risk stratification of viral infections. Disclosures Agathangelidis: Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Abbvie: Research Funding; Janssen: Honoraria, Research Funding.

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. Disclosures Agathangelidis: 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.

Recent evidence indicates that TAp63, the prevalent isoform of TP63 in Chronic Lymphocytic Leukemia (CLL), is implicated in disease pathogenesis. In CLL, TAp63 expression, modulated by both immune signaling and epigenetic modifications, promotes leukemic cell survival and homing to the bone marrow. In activated normal B cells, the TAp63 transcription factor binds the BCL2 gene, participating in an anti-apoptotic pathway (axis NF-κB/TAp63/BCL2) augmenting cell survival. In this study, we investigated the expression of TAp63 in a large cohort of CLL cases and its potential fluctuation during disease progression. Additionally, in order to further understand the pro-survival role of TAp63 in CLL, we interrogated at the molecular level the interplay betweenΤAp63 and BCL2. Initially, using RT-qPCR we quantified TAp63 mRNA expression in 166 CLL patients, consisting of 89 with unmutated IGHV genes (U-CLL) and 77 with mutated IGHV genes (M-CLL), prior to administration of treatment. Significantly higher TAp63 mRNA levels were observed in U-CLL vs M-CLL (FD=13.83, p We subsequently investigated links between TAp63 and BCL2 by measuring BCL2 mRNA levels in 56 U-CLL cases from the present cohort and found statistically significant correlation with the corresponding TAp63 mRNA levels (spearman rho=0.31, p=0.01). To validate this observation, we undertook functional studies in the MEC1 CLL cell line. Considering that MEC1 cells express high TAp63 mRNA levels, we generated a stable MEC1 cell line to inducibly downregulate TAp63, using CRISPR/dCas9-KRAB upon treatment with doxycycline (Dox). We used 2 different guide RNAs (sgRNAs; sgRNA1, sgRNA2) targeting 2 distinct regions of the endogenous TAp63 promoter. After 5 days of induction, the expression levels of both TAp63 and BCL2 were quantified by one step RT-qPCR in Tet-on-dCas9-KRAB-sgRNA-TAp63 MEC1 cells. Inducible downregulation of TAp63 expression (gRNA1: FD=1.7, gRNA2: FD=1.53) resulted in downregulation of BCL2 expression (gRNA1: FD=1.34, gRNA2: FD=1.12) with strong correlation (rho=0.97, p In conclusion, we provide evidence suggesting that up-regulated TAp63 expression represents a novel resistance mechanism to chemoimmunotherapy in CLL. The pro-survival role of TAp63 is supported by its strong association with BCL2. Indeed, based on the present findings, TAp63 appears to act as a positive modulator of BCL2 in CLL cells, rendering them less responsive to apoptosis induction with the BCL2 inhibitor Venetoclax. Disclosures Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

We and others recently reported mutations within the RPS15 gene, encoding a component of the 40S ribosomal subunit, in clinically aggressive chronic lymphocytic leukemia (CLL). RPS15 mutations resided within an evolutionary conserved region, alluding to an oncogenic rather than a tumor-suppressor role. Our pilot functional analysis revealed that, similar to other ribosomal proteins (RPs), RPS15 also binds MDM2 and may impact on the p53 response. Here, we performed ribosome profiling in order to gain global insight into changes in translation induced by RPS15 mutations in CLL cells. This technique involves measuring translational efficiency (TE), by comparing the levels of ribosome-associated mRNA footprints against the total mRNA for each gene. For 6 CLL cases bearing mutant (mut, n=3) or wildtype (wt, n=3) RPS15, we obtained both ribosome-protected footprints (RPFs) and matching mRNA sequencing data. In parallel, we created stable MEC1 CLL cell lines expressing an additional copy of wt or mut RPS15 (131S) by lentiviral transduction; validation of the transgene expression was performed by Sanger sequencing of amplified cDNAs. Ribosome footprinting and subsequent library preparation of RPFs and total mRNA for all samples was performed with the Illumina Truseq Ribo Profile Kit and all libraries were sequenced on a NextSeq500 instrument. Reads were aligned to the human hg19 genome using Bowtie2. SystemPipeR was used to determine the percentage of reads mapping to 5' UTRs, CDS, and 3' UTRs and triplet periodicity was assessed using RibORF. The RPFs were of high quality, as assessed by expected RPF size (28-30nt), CDS enrichment, and triplet periodicity. To determine differentially expressed genes between RPS15-mut vs RPS15-wt cases we used DESeq2 while, for differentially translated genes we used Xtail. Changes in transcription and translation between PRS15-wt vs RPS15-mut cases showed limited overlap in both primary CLL cells and cell lines (12.8% and 12.9%, respectively), indicating the potential of ribosome profiling to reveal additional information compared to RNA sequencing alone. In primary CLL cells, 474 genes showed differences only at the transcription level (log2FC mRNA>I1I, pI1I, pI1I), while 1859 genes were regulated only at the translation level (log2FC RPF>I1I). Overall, 771 genes displayed differential TE (log2FC TE Disclosures Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

Non-neoplastic lymphadenopathy (NNL) associated with the human immunodeficiency virus (HIV) infection may develop concurrently with the onset of HIV viremia (acute retroviral syndrome) that can persist beyond the acute phase. Histopathological findings at this early phase mainly pertain to hyperplastic changes with large lymphoid follicles; with time, the number of lymphoid follicles diminishes, while plasma cells increase; at the extreme is a pattern characterized by sclerosis of the germinal centers in the residual follicles. HIV-specific CD8+ T cell responses have been reported and certain viral protein epitopes have been identified e.g. the p24 protein, a component of the HIV particle capsid. Overall, these findings reflect an ongoing immune response that is still incompletely characterized at the molecular level, particularly as it concerns the composition of the T cell receptor (TR) gene repertoire. In order to obtain a comprehensive view into the role of antigen selection in shaping T cell responses in HIV-associated NNL [HIV(+) NNL], we studied in-depth the TR repertoire in: (i) lymph node biopsy samples from 12 patients with HIV(+) NNL, (ii) lymph node samples from 5 non-HIV patients with reactive lymphadenopathy [HIV(-) RL]; and, (iii) peripheral blood samples from 4 healthy, HIV-seronegative individuals without lymphadenopathy [healthy controls, HIV(-) HC]. Genomic DNA was isolated from either paraffin-embedded lymph nodes (for patients with lymphadenopathy) or blood mononuclear cells (for healthy individuals). TRBV-TRBD-TRBJ gene rearrangements were amplified according to the BIOMED2 protocol. PCR products were subjected to next generation sequencing (NGS) on the MiSeq Illumina Platform. NGS data analysis, interpretation and visualization was performed by a validated, in-house bioinformatics pipeline. Overall, we obtained: (i) 1,440,305 (mean: 120,025) productive rearrangement sequences in the HIV(+) NNL group; (ii) 702,533 (mean: 140,506) productive sequences in the HIV(-) RL group; and, (iii) 539,981 (mean: 134,995) productive sequences in HIV(-) HC cases. Rearrangements with identical TRBV gene usage and CDR3 sequence were defined as clonotypes. In total, we identified 15,553 unique clonotypes in patients with HIV(+) NNL (mean: 1,296, range: 337-6,212), 53,874 in HIV(-) RL (mean: 10,774, range: 3,336-16,304) and 220,069 clonotypes in HIV(-) HC cases (mean; 55,017, range: 35,430-68,916), indicating significant repertoire restriction in the former group. Indeed, this group was characterized by an increased level of oligoclonality compared to the other two groups: the mean values of the sum of relative frequencies for the 10 most frequent clonotypes were 80%, 19.6% and 16.5%, respectively. Seven of 12 HIV(+) NNL cases carried the same dominant clonotype (TRBV29-1, SVDPSGTGGEGYT) that was also found in the remaining 5 patients of this group, albeit at lower frequencies; in contrast, it was completely absent in the HIV(-) RL and HIV(-) HC groups. Regarding the TRBV gene repertoire, the TRBV29-1 gene was overrepresented (p Disclosures Gemenetzi: Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding.

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. Disclosures Gemenetzi: 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.

Genome surveys have offered a comprehensive view of the genetic landscape of chronic lymphocytic leukemia (CLL), identifying several recurrently mutated genes, including myeloid differentiation primary response 88 (MYD88). The predominant mutation concerns a p.L265P substitution within exon 5,1,2 which leads to constitutive nuclear factor kappaB stimulation, thus conferring a proliferation and survival advantage to the mutant cells.1 MYD88 mutations reach up to 2% to 5% in CLL and are strikingly enriched among patients expressing mutated IGHV genes (M-CLL).

Immunogenetic analysis of MM has proven instrumental in elucidating disease ontogeny e.g. by revealing the clonal relationship between switch variants expressed by the bone marrow plasma cells and myeloma progenitors in the marrow and blood; demonstrating the marked under-representation of the inherently autoreactive IGHV4-34 gene; and, identifying patterns of somatic hypermutation (SHM) indicative of post-germinal center derivation. Yet, limited information exists about the composition of the immunoglobulin (IG) gene repertoire in MM cases expressing different heavy chain isotype, in particular A versus G. This is relevant in light of studies showing an overall higher SHM impact in CD27+IgA+ compared to CD27+IgG+ normal memory B cells, perhaps reflecting a distinct location of the immune response, especially considering that IgA class switching mostly occurs in mucosa-associated lymphoid tissues. From a clinical perspective, it is also relevant to note that IgA patients exhibit a higher incidence of the t(4;14) translocation, shorter progression-free survival and worse median overall survival compared to IgG patients. Here, we explored potential differences in the immunoprofiles of IgA versus IgG MM focusing on IG gene repertoire and SHM characteristics. In total, 428 patients with a diagnosis of MM following the IMWG criteria from collaborating institutions in Greece, Italy and Spain (n=355) or retrieved from the LIGM-DB (n=73) were included in the study. Of these, 135 and 293 belonged to IgA and IgG MM groups, respectively. Amongst the evaluated productive IG rearrangements, IGHV3 subgroup genes predominated in both groups (IgA: 58.5%; IgG: 52.2%). However, at the individual gene level, major asymmetries were noted, since only 7 IGHV genes accounted for 41.6% of the IgA and 46.7% of the IgG cases, respectively. Of these, 3 genes were shared between IgA and IgG MM cases: IGHV3-30 (IgA: 11.9% - IgG: 13.3%), IGHV3-23 (IgA: 5.2% - IgG: 6.8%) and IGHV3-9 (IgA: 6.7% - IgG: 4.4%), whereas the remaining 4 of the 7 most frequent genes were specific for each group with significant (p Disclosures Terpos: Celgene: Honoraria; Novartis: Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; BMS: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Chronic lymphocytic leukemia (CLL) is characterized by remarkable skewing of the B-cell receptor immunoglobulin (BcR IG) repertoire, culminating in the existence of subsets of patients with stereotyped BcR IGs. This implies antigen selection in the natural history of CLL, ultimately affecting clonal behavior. Currently, limited information is available regarding the role of antigens in the selection and activation of cognate T cells, although this is relevant in light of B and T cell interactions inducing T cell tolerance. Our preliminary next-generation sequencing (NGS) studies in 11 patients assigned to stereotyped subset #4, a clinically indolent disease subgroup, indicated T-cell receptor beta chain (TRB) gene repertoire restriction and oligoclonality. Prompted by these observations, here we sought to obtain a comprehensive view of the T-cell repertoire in CLL by extending our analysis to 36 untreated CLL patients, either assigned to major stereotyped subsets [subset #4 (n=11), subset #1 (n=10), subset #2 (n=4), subset #16 (n=1)] or non-subset cases [with mutated-M (n=5) or unmutated-UM clonotypic BcR IGs (n=5)]. Starting material was PB mononuclear cells (n=27), purified CD4+ and CD8+ T-cell subpopulations (n=10), bone marrow (n=2) or lymph node (LN) tissue (n=1). Three patients were studied overtime. Multiple sample and PCR replicates, as well as 3 age-matched healthy controls were also included. TRBV-TRBD-TRBJ gene rearrangements were amplified on cDNA (BIOMED2 protocol) and subjected to paired-end NGS, designed to cover the complementarity determining region 3 (CDR3) twice/sequence. In order to further increase the accuracy of results, raw NGS reads were subjected to a purpose-built, bioinformatics algorithm, performing: (i) length and quality filtering of raw reads; (ii) merging of filtered-in paired reads via local alignment; (iii) length and quality filtering of stitched sequences. No base calls of Q-score Disclosures Stamatopoulos: Janssen Pharmaceuticals: Research Funding; Gilead Sciences: Research Funding.

B cells residing the marginal zone (MZ) provide a first line of defense against blood borne pathogens, producing the greater part of circulating natural antibodies conferring protection against infection. Dysregulated homeostasis and function of MZ B cells has been implicated in a wide range of B lymphoproliferations, encompassing the distinct MZ lymphomas recognized by the WHO Classification, the related provisional entities and even chronic lymphocytic leukemia (CLL), for which a MZ derivation has been proposed. Here, taking advantage of a large multi-institutional series, we aimed at obtaining insight into the ontogenetic relationship of MZ lymphoproliferations, related entities and CLL through cross-comparison of their B cell receptor immunoglobulin (BcR IG) gene repertoires. Our sequence dataset included 3660 unique IGHV-IGHD-IGHJ gene rearrangement sequences from our collaborative centers and/or public databases derived from: (1) MZ lymphomas: splenic (SMZL), n=379; nodal (NMZL), n=37; extranodal (ENMZL), n=95; (2) provisional entities of postulated MZ origin, including splenic diffuse red pulp lymphoma (SDRL, n=16) and clonal B cell lymphocytosis of MZ origin (CBL-MZ, n=60); (3) persistent polyclonal B cell lymphocytosis (PPBL), n=286 (from 2 cases); (4) MZ cells isolated from six spleen specimens free of neoplastic cells at histological inspection (non-malignant MZ), obtained at surgery for cancer, n=489; (5) autoimmune conditions, n=1243; (6) various types of normal B cells, n=1055. The most pronounced IG gene repertoire skewing was observed in SMZL with the IGHV1-2*04 gene accounting for 26% of cases. Restrictions, though less striking, were also identified in the other MZ lymphomas as well: (i) the IGHV4-34 gene predominated in NMZL (14.3%); and, (ii) the IGHV1-69 gene predominated in ENMZL (14.6%), albeit with significantly different distribution depending on the primary site of involvement, ranging from 38% in salivary ENMZL to 11% in gastric ENMZL to 4% in ocular adnexa ENMZL (p Disclosures Ghia: Janssen Pharmaceuticals: Research Funding.

The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with >90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p Disclosures Tausch: Gilead: Other: Travel support. Shanafelt:Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Polyphenon E Int'l: Research Funding; Hospira: Research Funding; Janssen: Research Funding; Pharmactckucs: Research Funding; Cephalon: Research Funding. Niemann:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy; Novartis: Other: Travel grant. Langerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). Hallek:Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Ghia:Janssen Pharmaceuticals: Research Funding.

The development of CD3+/CD8+/CD57+ cytotoxic cell expansions after allogeneic hematopoietic cell transplantation (allo-HCT) driven by antigenic stimulation, viral or associated with chronic graft-versus-host disease (cGvHD), has been suggested as related with favorable outcome. Rituximab, an anti-CD20 humanized monoclonal antibody, has been linked to the development of oligo- or even monoclonal expansions of CD3+/CD8+/CD57+ T-large granular lymphocytes (T-LGLs) that can manifest with neutropenia of delayed origin in relation to Rituximab administration. We have recently reported remarkable skewing of the T-cell receptor (TR) gene repertoire in two allo-HCT transplanted patients with delayed neutropenia associated with T-LGL expansions developing in a context of GvHD and Rituximab administration for EBV reactivation. Prompted by these preliminary findings, we here extend our immunogenetic studies of the TR repertoire in patients receiving Rituximab post allo-HCT. The study group was comprised of 9 patients (including the two previously reported) aged 14-50 years (median 41) who were subjected to myeloablative allo-HCT (4 from matched related, 3 from matched unrelated donors), haplo-identical transplantation (1) or Reduced Intensity Conditioning-allo-HCT from sibling donor (1), all for hematological malignancies. All patients received Rituximab consecutively between 2010-2013 either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. In all patients TR gene repertoire analysis was performed at least one year after the transplantation (range 12-72 months), when immune reconstitution normally would have been achieved, and 5-24 months after the first treatment with Rituximab. Each patient received a mean of 7 cycles of Rituximab (range, 1-14). TRBV-TRBD-TRDJ gene rearrangements were PCR-amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data was interpreted using the IMGT/V-QUEST tool. A total of 164 sequences were analyzed (9-25/case, median=18) revealing 106 productive TRBV-TRBD-TRBJ rearrangements. Among the 29 TRBV functional genes identified only three accounted for 48% of cases: (i) TRBV27*01 (25%), (ii) TRBV6-5*01 (13%), (iii) TRBV6-2*01 (10%). Of note, TRBV27*01 has been reported as the most frequent TRBV gene in Rituximab-related late-onset neutropenia in CLL. All cases were found to carry clusters of identical (>=2) rearrangements corresponding to clonotypes. In the majority of cases (5/9), 2-4 (median 3) immunodominant clonotypes accounted for over 30% of the analyzed sequences (frequency of immunodominant clonotype/case 13-40%). Lymphocyte subpopulation analysis by flow cytometry in 6 patients revealed T-LGL expansion. Samples from additional time points (spanning a period of 10 years), pre- and post- Rituximab, were studied in one patient. Analysis of 71 sequences demonstrated progressive expansion of a certain clonotype overtime, associated with the emergence of steroid-refractory autoimmune hemolytic anemia in a context of CD3+CD8+CD57+ lymphoproliferation. This particular clonotype dominated the repertoire by far, thus establishing a diagnosis of T-LGL leukemia. which, remarkably, proved to be of donor origin (97% and 30% donor chimerism in T lymphocytes and total hematopoeisis, respectively). No association of oligoclonality to stronger GvL effect could be found among the rest of the patients. However, a strong correlation with cGvHD (100% vs 25% among polyclonal cases) was identified. Late-onset neutropenia was documented in 4/9 patients, regardless of the composition of the repertoire i.e whether it was polyclonal or oligo(mono)clonal. In conclusion, we report frequent development of oligoclonal cytotoxic T-cell populations after Rituximab treatment post allo-HCT likely of multifactorial evidence. Direct evidence of the anti-leukemic effect of this phenomenon could not be provided, however, the observed association of oligoclonality with GvHD and the development of a possible “T-LGL leukemia vs leukemia” effect in one patient is noteworthy and merits further investigation. Finally, the observed skewing of the TR gene repertoire strongly implicates antigen selection in the development of cytotoxic T-cell expansions after allo-HCT. Disclosures No relevant conflicts of interest to declare.

Until quite recently, the prevailing view, adopted by the WHO 2008 Classification, was that mantle-cell lymphoma (MCL) originates from a peripheral B cell located within the inner mantle zone, an area comprised of naïve pre-germinal center (GC) type B cells. However, this notion has been challenged by molecular and functional evidence. Indeed, MCL is characterized by a skewed repertoire of immunoglobulin heavy variable (IGHV) genes and by some imprint of somatic hypermutation (SHM) in the clonotypic IGHV genes of the great majority (~70%) of cases, indicating antigen selection. Furthermore, both relapsed/refractory and treatment-naïve patients with MCL exhibit remarkable responses to B-cell receptor signaling inhibitors, strongly supporting a role for microenvironmental triggering in the natural history of MCL. In the present study, we sought to obtain additional insight into MCL ontogeny through a combined morphologic, immunohistochemical and immunogenetic analysis of 230 patients with a diagnosis of MCL according to the 2008 WHO Classification criteria. The study group included 139 nodal, 32 extranodal, 18 primary splenic MCLs as well as 41 bone marrow biopsies (BMB) infiltrated by MCL. Morphologically, 144/206 (70%) cases were ascribed to the common variety, while 48/206 (23.3%) and 14/206 (6.7%) were characterized as blastoid or pleomorphic variant, respectively. The immunohistochemical analysis (on paraffin sections) focused on CD27, DBA.44 and IRF4 (MUM1), markers not normally expressed by the naïve pre-germinal centre B-cell of the inner mantle zone. The results were as follows: (i) 117/214 (54.7%) cases positive for CD27 expression; (ii) 18/176 (10.2%) cases positive for DBA.44; (iii) 53/98 (54%) cases positive for IRF4. Amongst CD27+ cases, 10/86 (11.6%) were also positive for DBA.44, whereas 27/51 (52.9%) were also positive for IRF4. Immunogenetic information regarding IGHV-IGHD-IGHJ gene rearrangements was available for 167 cases of the study. Fifty of 167 cases (30%) carried IGHV genes with no SHM (100% identity to the germline, GI), whereas the remaining 117 cases (70%) bore some imprint of SHM: in particular, 95/167 cases (56.8%) carried IGHV genes with 97-99.9% GI, while 22/167 cases (13.2%) carried IGHV genes with Disclosures Stamatopoulos: Janssen Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The role of antigen(s) in shaping the T-cell repertoire in chronic lymphocytic leukemia (CLL) is largely unexplored, though highly relevant in light of the interactions of the CLL B cells with T cells, effectively inducing tolerance to the latter. Our recent classic subcloning/Sanger sequencing studies of the T-cell receptor beta chain (TRB) gene repertoire in CLL indicated repertoire restriction, pointing to antigenic selection. However, due to the inherent limitations of low-throughput analysis, definitive conclusions were not possible. Here, we sought to advance the analytical depth of our approach by employing high-throughput, next generation sequencing (NGS) for exploring the TRB gene repertoire in CLL. Our study included 9 untreated CLL cases assigned to two paradigmatic stereotyped subsets, namely clinically indolent subset #4 (n=7) and clinically aggressive subset #1 (n=2). RNA was isolated from peripheral blood mononuclear cells (n=7 cases) or purified CD4+ and CD8+ T cells (n=2, both subset #4). TRBV-TRBD-TRBJ gene rearrangements were amplified on cDNA according to the BIOMED2 protocol and were subjected to NGS (MiSeq Illumina Platform). The paired-end Illumina protocol allowed sequencing of the complementarity determining region 3 (CDR3) twice/read, thus increasing the accuracy of results. Still, considering the inherent limitations of PCR-based NGS, the experimental setup included many internal controls: (i) replicate samples of the same patient at the same timepoint; (ii) samples of the same patient at sequential time points (two-timepoint longitutinal analysis for 1 case); (iii) replicate cDNA samples for PCR amplification; and, (iv) analysis of a healthy individual. A bioinformatics pipeline was developed for raw NGS data processing, performing: (i) quality filtering of reads; (ii) merging of paired-end reads via local alignment; (iii) preparation of filtered-in fasta sequences for submission to the IMGT/HighV-QUEST tool; and, (iv) IMGT/HighV-QUEST metadata clustering, analysis and interpretation. Overall, 19 samples were analyzed, producing 7,920,136 TRBV-TRBD-TRBJ reads (median 359,957 reads/sample, median Q-score 38.3). Poor quality, incomplete, out-of-frame and unproductive rearrangements were filtered out (median 2.1% of reads/sample). For repertoire analyses, clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid CDR3 sequence) rather than single rearrangement reads were considered, so as to avoid possible biases due to clonal expansion following antigenic stimulation (median 56194 distinct clonotypes/sample, 33619 singletons versus 13725 expanded). Among the 53 functional TRBV genes identified, the following 5 predominated: TRBV12-3/12-4 (7.5%), TRBV19 (6.1%), TRBV5-1 (5.2%), TRBV29-1 (4.9%) and TRBV27 (4.8%), collectively accounting for 28.5% of the TRBV repertoire. Comparison of the TRBV gene repertoire of CD8+ vs CD4+ cells in subset #4 CLL cases showed that TRBV19 was overrepresented in the CD4+ compartment (9.4% versus 6.9%, p Disclosures No relevant conflicts of interest to declare.

The human IGHV4-34 gene encodes antibodies which are intrinsically autoreactive when the VH domain is unmutated. Therefore, B cells expressing IGHV4-34 B-cell receptor immunoglobulins (BcR IG) are normally under close scrutiny in order to avoid unwanted autoreactivity, especially against DNA. The IGHV4-34 gene is frequently utilized in chronic lymphocytic leukemia (CLL), where, typically, it shows a high load of somatic hypermutation (SHM). We have previously reported distinctive SHM patterns amongst IGHV4-34 CLL, especially for subsets with stereotyped BcR IG. However, although a large number of cases (~2000) was previously studied, since even the largest subsets account for only ~3% of CLL, meaningful conclusions could not be reached for smaller subsets. Here we revisit this issue in a series of 16,528 CLL cases and focus on IGHV4-34 expressing subsets: #4 (IGHV4-34/IGHD5-18/IGHJ6 | 156 cases, 0.9%); #11 (IGHV4-34/IGHD3-10/IGHJ4 | 16 cases, 0.1%); #16 (IGHV4-34/IGHD2-15/IGHJ6 | 41 cases, 0.25%); #29 (IGHV4-34/IGHD: unassignable/IGHJ3 | 39 cases, 0.24%); and #201 (IGHV4-34/IGHD: unassignable/IGHJ3 | 43 cases 0.26%). Focusing on codons 27-104 within the VH domain (from CDR1-IMGT to FR3-IMGT), we calculated the sequence distance between subsets and the corresponding IGHV4-34 germline sequence based on a pairwise qualitative and quantitative comparison of the respective amino acid composition. The minimum distance calculated, and hence the greatest identity, was observed between subsets #4 and #16, both concerning IgG-switched cases (IgG-CLL), which is notable given the overall rarity of IgG-CLL. In contrast, the maximum distance, implying the least identity, was between subsets #16 and #201, the latter concerning IgM/D-CLL. Extreme variations between subsets were noted in codons spanning the entire VH domain. This result is consistent with our finding of a subset-biased distribution of mutations over the VH domain. More specifically, while subsets #11, #16, #29 and #201 had a lower frequency of mutations within VH CDR1 compared to VH CDR2, the exact opposite was seen in subset #4, with 40% of mutations in VH CDR1 versus 27% in VH CDR2. In addition, subsets #4, #11, #16 and #29 had a similar distribution of mutations in VH FR2 and VH FR3, in contrast to subset #201 that showed a preference for VH FR3 over VH FR2. Consequently, we noted that certain positions were targeted in a subset-specific manner e.g. codon 28 in VH CDR1 was heavily targeted in subsets #4 (68.6%) and #16 (87.8%), with most cases carrying an acidic amino acid (AA) introduced by SHM, glycine to glutamic acid, G>E: 51.3% for subset #4 and 78% for subset #16. The high prevalence of acidic AA introduced by SHM in these subsets is notable considering the electropositive nature of their VH CDR3 (especially of subset #4), strongly recalling edited anti-DNA antibodies. Interestingly, the G>E change was identified at a much lower frequency in other IGHV4-34 subsets: 18.75% for subset #11; 2.6% for subset #29; 7% for subset #201, all of which carried electronegative VH CDR3. Further, we noted that certain positions were heavily targeted in all subsets e.g. 56-86% targeting for SHM at codon 92 in VH FR3 where serine is encoded by the agc triplet, the ”hottest of hotspots”. This result could be viewed as sequence- rather than subset-dependent and linked to the molecular features of this codon, which is supported by the low targeting of codon 93 (0-6%), also encoding serine by the tct triplet. Other positions were targeted in all subsets but at vastly different frequencies e.g. codon 64 was targeted in 37.8% in subset #4 rising to 100% in subset #29. Finally, positions heavily targeted by SHM in certain subsets were unmutated in other subsets e.g. codon 36 in VH CDR1 remained unmutated in subset #16, in contrast 76.9% of subset #29 were mutated at this position resulting in an AA change. In conclusion, we document different spectra of SHM and AA changes between stereotyped IGHV4-34 CLL subsets. The finding of subset-biased, recurrent AA changes at certain codons indicates that the respective progenitor cells may have responded in a specific manner to the selecting antigen(s), despite expressing the same IGHV gene, indicating a functional purpose for these modifications. This is exemplified by the molecular characteristics of the recurrent AA changes in subset #4, thereby offering interesting pathogenetic hints. Disclosures No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) is characterized by progressive hypogammaglobulinemia that can affect one or more immunoglobulin subclasses. Although many underlying mechanisms have been suggested, the pathogenesis of this phenomenon remains to be elucidated. In the present study, we revisit hypogammaglobulinemia in CLL through a combined clinicobiological approach aiming at identifying associations with particular disease profiles that would offer pathogenetic insight and guidance for further research. The study group included 412 CLL patients with available information about serum immunoglobulins either at diagnosis (n=380) or before treatment initiation (n=32). Patient characteristics were as follows: median age: 65 years; males/females: 266/146; Binet stage A: 272/335, unmutated IGHV genes (U-CLL): 140/412 cases (34%); CD38 expression: 59/330 cases (18%); clonotypic IG of the MD or G isotype: 250 and 43 cases, respectively; isolated del(13q): 64/136 (47%); trisomy 12: 18/183 (10%); del(11q): 18/186 (10%); del(17p): 11/189 (6%); NOTCH1 del7544-45/p.P2514Rfs*4: 8/219 (4%). With a median follow up of 5 years, 152/329 cases (46%) received treatment. Decreased immunoglobulin serum levels in at least one subclass were identified in 220/412 patients (53%), as follows: (i) decreased IgM, 172/412 cases (41%); (ii) decreased IgG, 78/412 cases (19%); (iii) decreased IgA, 100/412 cases (24%). In 36/412 cases (9%), a decrease in all serum immunoglobulin subclasses was noted. No statistically significant differences were identified between patients with normal serum immunoglobulin levels versus those with hypogammaglobulinemia regarding age, gender, disease burden at diagnosis, IGHV gene mutational status, CD38 expression, cytogenetic aberrations, NOTCH1 mutations and the incidence of a second malignancy. However patients with hypogammaglobulinemia exhibited increased need for treatment compared to patients with normal serum immunoglobulins (91/175 vs 61/154 respectively, p=0.025). Among cases with hypogammaglobulinemia, 90 (41%) and 26 (12%) exhibited isolated IgM and IgA subclass deficiency, respectively; isolated IgG decrease, was relatively rare (10/220 cases, 4%). Interestingly, when comparing isolated IgA versus other subclass deficiencies, statistically significant associations were identified with (i) advanced clinical stage (Binet B/C, Rai III/IV) (p=0.002); (ii) female gender (p=0.041); and, (iii) NOTCH1 mutations (p=0.004). A propos of the latter, it is noteworthy that in 5/8 (63%) mutant NOTCH1 cases with hypogammaglobulinemia, the affected subclass was IgA. Within our cohort, we identified cases belonging to one of three different, well characterized subsets with stereotyped B-cell receptor immunoglobulin (BcR IG), namely: (1) subset #1 (clan I IGHV genes/IGKV1(D)-39): U-CLL, clinically aggressive, n=12; (2) subset #2 (IGHV3-21/IGLV3-21), mixed IGHV mutational status, noted clinical aggressiveness, n=5; and, (3) subset #4, mutated IGHV4-34/IGKV2-30 BcR IG, clinically indolent, n=12. Notably, all subset #2 cases showed low levels of at least one serum subclass, while in 4/5 and 3/5 cases, two or all three immunoglobulin subclasses were affected. Although numbers are small, the incidence of hypogammaglobulinemia in subset #2 was significantly (p Disclosures No relevant conflicts of interest to declare.

Preliminary observations from essentially small patient series indicate that certain recurrent gene mutations may be enriched in subsets of chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptors (BcR). On these grounds, it could be argued that differential modes of immune signaling, in the context of subset-biased antigen-immunoglobulin (IG) interactions, may be associated with the acquisition and/or selection of certain genomic aberrations within various stereotyped CLL subsets. With this in mind, we here sought to explore the genetic background of 10 major stereotyped subsets which collectively account for ~11% of all CLL and represent both IGHV unmutated (U-CLL) and/or mutated (M-CLL) cases. We focused on recurrent mutations within the NOTCH1 (entire exon 34 or targeted analysis for del7544-45), TP53 (exons 4-9), SF3B1 (exons 14-16), BIRC3 (exons 6-9) and MYD88 (exon 5) genes. Overall, 647 cases were analyzed, belonging to the following major subsets: (i) U-CLL: #1 (the largest within U-CLL, clinically aggressive), n=139; #3, n=39; #5, n=22; #6, n=48; #7, n=74; #8, n=46; #59, n=19 and #99, n=18; (ii) M-CLL: #4 (the largest within M-CLL, particularly indolent), n=78; and, (iii) subset #2 (the largest overall, variable mutational status and clinically aggressive), n=164. All cases were devoid of MYD88 mutations, which was not surprising given that our cohort was predominantly composed of U-CLL. Mutations within the BIRC3 gene were either absent (#2, #4, #6 and #59) or rare (#1, #3, #5, #7, #8 and #99; frequency 1.5%-7%) with no clear bias to any subset. BIRC3-mutant cases frequently co-existed with either del(11q) or trisomy 12. NOTCH1 mutations were more frequent in subsets #1, #6, #8, #59 and #99 (frequency, 22%-32%), sharply contrasting subsets #2 or #3 (4% and 7%, respectively) (p Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The chronic lymphocytic leukemia (CLL) immunoglobulin (IG) heavy chain repertoire is known to display biased immunoglobulin variable heavy-chain (IGHV) gene usage, remarkable complementarity determining region 3 (HCDR3) stereotypy as well as distinctive somatic hypermutation (SHM) patterns, at least for subsets of cases. Our aim in the present study was to similarly investigate the IG light chain (LC) genes in terms of mutation frequency and targeting and CDR3 stereotypy to elucidate if the LC may play a significant complementary role in antigen recognition in CLL. We thus examined SHM patterns and secondary rearrangements of the IG LC gene loci in a total of 612 IGKV-J and 279 IGLV-J rearrangements from 725 patients with CLL. Firstly, we observed a highly restricted light chain gene usage in the vast majority of CLL cases with stereotyped HCDR3s. In particular, stereotyped IGHV3-21 CLL cases were characterized by a strikingly biased expression of lambda light chains utilizing the IGLV3-21 gene (36/37 cases of subset#2), whereas all 15 subset #4 cases with stereotyped IGHV4-34 IGs carried an IGKV2-30 rearrangement. In addition, subset-biased light chain CDR3 motifs were identified in groups of sequences utilizing the same IGKV or IGLV gene. For example, all 30 IGKV1-39/1D-39 light chains of subset#1 (using stereotyped IGHV1/5/7 genes) carried notably long KCDR3s (10–11 amino acids) generated by significant N region addition and characterized by the frequent introduction of a junctional proline (26/30 cases). Important differences regarding mutational load were observed in groups of sequences utilizing the same IGKV or IGLV gene and/or belonging to subsets with stereotyped B cell receptors (BCRs). In fact, significant differences were observed with regard to mutational status among groups of sequences utilizing different alleles of certain IGK/LV genes (specifically the IGKV1-5, IGLV1-51 and IGLV3-21 genes). At cohort level, the SHM patterns were typical of a canonical SHM process. A clustering of R mutations in KCDR1 was evident for all IGKV subgroups with the notable exception of the IGKV2 subgroup, which exhibited preferential targeting to the KCDR2, especially in IGKV2-30 rearrangements of cases with stereotyped IGHV4-34/IGKV2-30 BCRs (subset#4). Recurrent amino acid changes at certain positions across the entire IGKV/IGLV sequence were observed at a high frequency (27–67% of cases) in a number of stereotyped subsets, especially those expressing the IGHV3-21/IGLV3-21 BCR (subset #2) and the IGHV4-34/IGKV2-30 BCR (subset #4). Comparison with CLL LC sequences carrying heterogeneous K/LCDR3s or non-CLL LC sequences revealed that these distinct amino acid changes are greatly under-represented in such groups and appear therefore to be “subset-biased”. Finally, a significant proportion of CLL cases (63 cases; 26 kappa- and 37 lambda-expressing) with monotypic LC expression were found to carry multiple potentially functional LC rearrangements. Of note, nineteen of these 63 cases (30%) belonged to subsets with stereotyped BCRs. This finding alludes to the possibility of secondary rearrangements most likely occurring in the context of (auto)antigen-driven receptor editing, particularly in the case of stereotyped subsets. In conclusion, SHM targeting in CLL LCs appears to be just as precise and, most likely, functionally driven as in heavy chains. Secondary LC gene rearrangements and subset-biased mutations in CLL LC genes are strong indications that LCs are crucial in shaping the specificity of leukemic BCRs, in association with defined heavy chains. Therefore, CLL is characterized not only by stereotyped HCDR3 and heavy chains but, rather, by stereotyped BCRs involving both chains, which create distinctive antigen binding grooves.