ALBERT

Description: Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B cell lymphocytosis (MBL), characterized by the presence of monoclonal CLL-like B cells in the peripheral blood, yet at lower numbers than those required for the diagnosis of CLL. MBL is distinguished into low-count (LC-MBL) and high-count (HC-MBL), based on the number of circulating CLL-like cells. While the former does not virtually progress into a clinically relevant disease, the latter may evolve into CLL at a rate of 1% per year. In CLL, genomic studies have led to the discovery of recurrent gene mutations that drive disease progression. These driver mutations may be detected in HC-MBL and even in multipotent hematopoietic progenitor cells from CLL patients, suggesting that they may be essential for CLL onset. Using whole-genome sequencing (WGS) we profiled LC-MBL and HC-MBL cases but also CLL patients with stable lymphocytosis (range: 39.8-81.8*109 CLL cells/l) for 〉10 years (hereafter termed indolent CLL). This would refine our understanding of the type of genetic aberrations that may be involved in the initial transformation rather than linked to clinical progression as is the case for most, if not all, CLL driver mutations. To this end, we whole-genome sequenced CD19+CD5+CD20dim cells from 6 LC-MBL, 5 HC-MBL and 5 indolent CLL cases; buccal control DNA and polymorphonuclear (PMN) cells were analysed in all cases. We also performed targeted deep-sequencing on 11 known driver genes (ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, FBXW7) in 8 LC-MBL, 13 HC-MBL and 7 indolent CLL cases and paired PMN samples. Overall similar mutation signatures/frequencies were observed for LC/HC-MBL and CLL concerning i) the entire genome; with an average of 2040 somatic mutations observed for LC-MBL, 2558 for HC-MBL and 2400 for CLL (186 for PMN samples), as well as ii) in the exome; with an average of non-synonymous mutations of 8.9 for LC-MBL, 14.6 for HC-MBL, 11.6 for indolent CLL (0.9 for PMN samples). Regarding putative CLL driver genes, WGS analysis revealed only 2 somatic mutations within NOTCH1, and FBXW7 in one HC-MBL case each. After stringent filtering, 106 non-coding variants (NCVs) of potential relevance to CLL were identified in all MBL/CLL samples and 4 NCVs in 2/24 PMN samples. Seventy-two of 110 NCVs (65.5%) caused a potential breaking event in transcription factor binding motifs (TFBM). Of these, 29 concerned cancer-associated genes, including BTG2, BCL6 and BIRC3 (4, 2 and 2 samples, respectively), while 16 concerned genes implicated in pathways critical for CLL e.g. the NF-κB and spliceosome pathways. Shared mutations between MBL/CLL and their paired PMN samples were identified in all cases: 2 mutations were located within exons, whereas an average of 15.8 mutations/case for LC-MBL, 8.2 for HC-MBL and 9 for CLL, respectively, concerned the non-coding part. Finally, 16 sCNAs were identified in 9 MBL/CLL samples; of the Döhner model aberrations, only del(13q) was detected in 7/9 cases bearing sCNAs (2 LC-MBL, 3 HC-MBL, 2 indolent CLL). Targeted deep-sequencing analysis (coverage 3000x) confirmed the 2 variants detected by WGS, i.e. in NOTCH1 (n=1) and FBXW7 (n=1), while 4 subclonal likely damaging variants were detected with a VAF 10 years display similar low genomic complexity and absence of exonic driver mutations, assessed both with WGS and deep-sequencing, underscoring their common low propensity to progress. On the other hand, HC-MBL comprising cases that may ultimately evolve into clinically relevant CLL can acquire exonic driver mutations associated with more dismal prognosis, as exemplified by subclonal driver mutations detected by deep-sequenicng. The existence of NCVs in TFBMs targeting pathways critical for CLL prompts further investigation into their actual relevance to the clinical behavior. Shared mutations between CLL and PMN cells indicate that some somatic mutations may occur before CLL onset, likely at the hematopoietic stem-cell level. Their potential oncogenic role likely depends on the cellular context and/or microenvironmental stimuli to which the affected cells are exposed. Disclosures Stamatopoulos: Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses. Ghia:Adaptive: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding.

Description: Chronic lymphocytic leukemia (CLL) leukemic cells express B-cell receptor immunoglobulin (BcR IG) whose signaling is of paramount importance throughout the natural history of the disease. Indeed, signaling pathways downstream of the BcR are constitutively active in all cases of CLL and inhibitors of the Bruton's tyrosine kinase BTK (Ibrutinib) or PI3Kδ (Idelalisib), two downstream signaling effectors, are clinically effective. This functional evidence complements earlier molecular observations supporting antigen drive in CLL ontogeny, including the distinction of CLL into cases with somatically hypermutated BcR IG (M-CLL) that have a significantly better outcome compared to those with unmutated, germline-like receptors (U-CLL). CLL also displays a remarkably skewed BcR IG gene repertoire, culminating in the existence of highly homologous, stereotyped BcR IG in 〉30% of cases, indicating selection by a limited set of antigenis. A number of potential antigenic elements have been described, being recognized by the monoclonal receptors and able to deliver intracellular signals. More recently, it has been reported that CLL cells are endowed with the apparently unique property of autonomous signaling, since individual CLL-derived BcR IG can promote Ca2+ influx and NF-κB target gene transcription in a reconstituted B cell system upon self-recognition of common BcR-intrinsic epitopes. However, the precise molecular details of such process are unknown. In order to gain insight into the molecular interactions, particularly to further understand the role played by autonomous signaling, we determined the crystal structures of two BcR IG of CLL cases assigned to subset #4. This is a CLL subset expressing stereotyped, G(κ)-switched BcR IG encoded by the IGHV4-34/IGKV2-30 gene combination. Subset #4 accounts for ~1% of all CLL and is the largest within M-CLL, distinctive for a particularly indolent clinical course. BcR IG derived from two subset #4 cases were found to bind autologously via their VH CDR3 loops to a composite surface spanning the variable and constant regions of the heavy chain; the relevant epitope is conserved in all cases belonging to subset #4 and differs from other non-subset #4 BcR IG. This specific self-recognition was identified as dependent on the individual IG gene usage in the BcR, and is functionally relevant as it occurs in solution and leads to intracellular signalling in B cells. Analysis of epitope and paratope mutants revealed that the interactions observed in the crystal structures are mediated by a few critical amino acid residues. Indeed, the distinctively conserved amino acid residues in the VH CDR3 loop of the BcR IG both dictate a specific VH-VK pairing and shape the combining site for autologous recognition. Moreover, the epitope comprises specific amino acids from the CH1 domain that restrict the autologous recognition to IgG molecules. Finally, we found persisting long-lived interaction occurring between subset #4 BcR IGs, thus recalling high affinity receptor-cognate antigen interactions associated with the induction of anergy. This scenario well fits with the anergic phenotype of the subset #4 leukemic cells, and thus provides a biochemical explanation for the indolent clinical course of this subset. In conclusion, though focusing on a particular CLL subset, the structural and biochemical analysis here presented describes a general model for autologous recognition that may epitomize the molecular events leading to the expansion of CLL B lymphocytes at large. It is conceivable that CLL-associated BcR IGs can each bind to a distinct internal epitope with the specific nature of the interaction dictated by diverse factors e.g. VDJ recombination, heavy and light chain pairing, SHM, and isotype switch. The strength and persistence of the autologous recognition can then lead to a specific outcome in the intracellular signaling process, ranging from proliferation to anergy. The structural diversity thus produced in the BcR IG development may be linked to and underlie the heterogeneity characterizing CLL at the biological and clinical level. Disclosures Stamatopoulos: Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding. Ghia:Pharmacyclics: Honoraria; Gilead: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria; Roche: Research Funding; GSK: Research Funding; AbbVie: Honoraria; Celgene: Honoraria; Adaptive Biotechnologies: Consultancy.

Description: Abstract 2240 Poster Board II-217 Molecular monitoring of the CMV viral load in the blood after allogeneic hematopoietic cell transplantation (allo-HCT) by quantitative real-time polymerase chain reaction (RQ-PCR) assays is considered as the most important measure for CMV disease prevention and may prompt the initiation of preemptive therapy. Molecular assays have a high negative predictive value, yet their precise role in the establishment of CMV infection in biological specimens other than plasma has not been defined conclusively. Furthermore, several technical aspects remain unresolved, in particular the use of cut-offs for positivity, given that, generally, viral loads (viral genome copies, VGC) less than 0,5-2,5log10 cannot provide linearity in the results. We retrospectively evaluated the clinical significance of positive RQ-PCR tests for CMV DNA in biological fluids other than plasma from 73 patients after allo-HCT, with a special emphasis on samples with a low viral load (

Description: Background: B cell receptor (BCR) signaling is a central pathway in Chronic Lymphocytic Leukemia (CLL) pathogenesis that is activated by interactions between CLL cells and the microenvironment in secondary lymphoid organs. Nurselike cells (NLCs) are an important component of this microenvironment, and co-culture of CLL cells with NLCs activates BCR signaling. CLL BCRs are able to recognize vimentin and calreticulin proteins exposed on the surface of NLCs and these interactions are responsible for stromal-mediated anti-apoptotic effects. However, the exact mechanism of BCR activation and the nature of the BCR ligands expressed by NLCs still remain incompletely defined. Aim: The aim of this project is to identify and validate ligands expressed by NLCs that activate BCRs on CLL cells. Methods: CLL PBMCs from 3 CLL patients were cultured in vitro for 14 days until outgrowth of NLCs. Then, NLCs were harvested and lysed, followed by immunoprecipitation with recombinant monoclonal antibodies obtained from 4 different CLL patients carrying unmutated IGHV genes (U-CLL). Immunoprecipitation of human hTERT mesenchymal stromal cells was used as a negative control. Immunoprecipitated proteins were analyzed by label-free quantitative mass spectrometry followed by bioinformatic data analysis using the softwares MaxQuant and Perseus. The quantitative mass spectrometric data enabled us to distinguish between unspecific background proteins and putative BCR ligands. Results: In all samples, around 2600 proteins were identified and around 2000 of them were quantified using mass spectrometry. Unsupervised hierarchical clustering identified the enrichment patterns of NLC-derived BCR ligands. We identified 6 different protein clusters; among them, one cluster included 11 putative CLL BCR antigens with a fold-change cut-off above 10, which were enriched in all 3 NLC samples, but not in hTERT cells. These BCR ligands included cytoskeletal proteins, ER-associated proteins, and membrane-associated proteins, some of them with known auto-antigenic function in other diseases. Conclusion: Recombinant BCRs from U-CLL patients recognize a large number of proteins expressed by NLCs, identified through immunoprecipitation of NLC lysates with CLL BCRs, followed by label-free mass spectrometry. The identified ligands will be further validated by epitope-mapping and BCR activation functional studies to allow a better characterization of the pathogenic antigens in CLL, and of the mechanisms driving CLL survival in the tissue microenvironment. Disclosures Wierda: Glaxo-Smith-Kline Inc.: Research Funding; Celgene Corp.: Consultancy. Estrov:incyte: Consultancy, Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Description: Abstract 1775 The immunoglobulin gene repertoire in CLL is remarkably restricted with greater than 30% of cases carrying quasi-identical (stereotyped)heavy complementarity-determining region 3 (VH CDR3) sequences. Indeed, cases can be clustered into different subsets based on shared, subset-biased motifs within the clonotypic VH CDR3s, with, notably, only a handful of subsets accounting for almost 10% of all CLL. VH CDR3 stereotypes are more frequent in cases with unmutated IGHV genes (U-CLL) who are associated with adverse prognosis. In principle, VH CDR3 stereotypy might allow to exploit these IG motifs as candidate Tumor Associated Antigens (TAA) for targeted immunotherapy of CLL. The aim of our study was to validate as potential TAA subset-specific IG motifs from major CLL subsets, focusing especially on subsets #1 and #2 that are the largest overall and both associated with aggressive clinical course. We have so far identified, by in silico analysis, 1–3 long peptides (15-mer) encompassing the VH CDR3 protein regions of subsets #1, 2, 4, 6, 8, 10 with (i) high binding score to MHC class II molecules and (ii) also containing minimal HLA class I-specific epitopes (HLA-A2, -A3, -A24, DR1, DR7, DR13 that are most frequent in the Caucasian population). Blood lymphocytes from 18 CLL patients were collected and phenotyped by flow cytometry with appropriate antibodies to assess the expression of stimulatory, co-stimulatory and negative regulatory molecules on both T and B cells. In addition, HLA typing of CLL patients was performed to select patients expressing the aforementioned HLA molecules. Overall, 13/18 patients matched the defined HLA class I and/or class II molecules. Negatively purified T cells from 11 CLL patients expressing HLA-A2 and/or DR13 have been then stimulated in vitro with the synthesized peptides of the specific stereotype (subset #1 and 2) in the presence of culture medium containing 5% of human serum plus IL-2 (20 IU/ml) and IL-15 (10 ng/ml). These T lymphocytes were then weekly stimulated with autologous irradiated antigen presenting cells (APC; monocytes, B cells, etc.) pulsed with the peptides. Starting from the third week of culture, the specific recognition of CDR3-derived TAAs and of tumor cells (autologous CLL cells) by the T cell cultures has been assessed by in vitro functional assays (ELISPOT assay). We were able to isolate CDR3- (subsets #1 and #2) and tumor-specific T cells from 5/11 CLL patients. In addition, in 4 selected patients the Ag- and tumor specific T lymphocytes have been expanded in vitro by Rapid Expansion Protocol (REP), based on the stimulation of T cells with allogeneic irradiated PBMCs from healthy donors plus OKT3 and high doses of IL-2. Using this protocol we were able to obtain large numbers (2–10 ×109) of anti-CDR3 T cells in all 4 cases tested, thereby, in principle, achieving the potential to use this protocol for expanding sufficient cells for clinical applications. Interestingly, post-REP T cell cultures showed enrichment (85–90%) of CD3+CD8+ T cells and down-modulation of negative regulatory molecules, such as CTLA-4, as compared to pre-REP in vitro stimulated T cells. These cells could be expanded in vitro for up to 6 weeks without any decay in proliferation. Taken together, these results indicate that stereotyped VH CDR3 peptide sequences can represent candidate antigens to elicit T cell-mediated anti-CLL responses, especially in poor prognosis cases, where therapeutic innovation is more urgently needed. After validation of this protocol in a larger series, our results may provide the proof of principle for the design of new immunotherapy protocols for CLL, including both active vaccination and adoptive cell therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Mounting evidence suggests that specific modalities of B-cell receptor (BcR) and Toll-like receptor (TLR) collaboration and/or regulation may exist in chronic lymphocytic leukemia (CLL), eventually impacting on the behavior of the malignant clones. CLL patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BcRs, SS4), the largest subset within mutated CLL, are characterized clinically by early age at diagnosis and indolent disease course and genetically by few lesions, with deletion of chromosome 13q representing the exclusive recurrent aberration. Examination of the IG sequences of SS4 clonotypic BcRs allows for parallels to be drawn with pathogenic anti-DNA antibodies since both carry long positively charged VH CDR3s. However, SS4 IGs also display distinctive somatic hypermutation (SHM) patterns, the most notable concerning the obligatory introduction of negatively charged residues in either the heavy or the light chains or both, alluding to editing of an autoreactive progenitor. Moreover, SS4 BcRs continue to acquire SHMs within their IG genes overtime, implying antigen selection in the clonal evolution of CLL SS4, albeit without a negative clinical impact. Altogether, these findings suggest that CLL SS4 cells may exist in an anergic state showing attenuated responses to selecting (auto)antigenic elements. To test this hypothesis, after BcR engagement in vitro we analysed basal phosphorylated ERK (pERK) levels and calcium mobilization, both known to be features of B-cell anergic state. Nine of 10 SS4 cases expressed high pERK levels and were unable to increase intracellular Ca2+ upon BcR crosslinking; moreover, stimulation through the BcR had no effect on pERK levels. Primary cell culture for 50min caused a statistically significant reduction of pERK, while culture for 6-24 hours restored the ability of the cells to respond to BcR activation by inducing ERK phosphorylation. Furthermore, analysis of serial samples of 4 SS4 cases over a period spanning 5-7 years revealed consistently positive pERK expression. On these grounds, we conclude that SS4 CLL clones are anergic through the BcR possibly because of chronic (auto)antigen activation. To determine if this profile is SS4-biased or linked to the usage of the IGHV4-34 gene, we also performed similar experiments in 6 cases using this gene in mutated non-stereotyped rearrangements of the MD isotype and found a more heterogeneous pattern: 3/4 cases were pERK negative, while 3/6 cases showed Ca2+ fluxes after BcR crosslinking. Given that SS4 cells are responsive to TLR1/2 ligands, we next investigated if TLR signals can modulate BcR anergy. To this end, SS4 CLL cells pre-stimulated through TLR1/2 with Pam3CSK4 for 50min were then subjected to BcR crosslinking for 10min. TLR1/2 stimulation up-regulated pERK levels, confirming our previous finding that CLL SS4 cells are responsive to TLR1/2 signalling. Notably, BcR crosslinking in TLR1/2-pretreated cells induced even higher pERK levels, indicating that TLR1/2 triggering restores BcR functionality, likely breaching the anergic state. We have previously demonstrated that TLR1/2 stimulation induces up-regulation of the miR-17∼92 cluster eventually leading to down-regulation of critical BcR and/or TLR signaling molecules (i.e. MAP3K1, MAP2K3, MAPK8, CASP8, IKBKB) which are potential targets of these miRNAs. In order to experimentally validate the predicted mRNA-miRNA interactions, we performed 3’ UTR luciferase reporter assay experiments in HeLa cells and documented 5 novel interactions between miR-17∼92 cluster members and MAP3K1, MAP2K3 and MAPK8. To ensure that these interactions are valid also in CLL, we performed transfection experiments with miRNA mimics in negatively selected CLL B cells and obtained identical results. In conclusion, we demonstrate that SS4 CLL cells are anergic through the BcR and that stimulation through TLR1/2 may break B-cell anergy. Moreover, we propose a regulatory mechanism whereby TLR1/2 signals induce miR-17∼92 cluster up-regulation, leading to down-regulation of MAP3K1, MAP2K3 and MAPK8 and eventual modulation of ERK phosphorylation, a key to the anergic state. On a broader scale, the detailed molecular and biochemical characterization of immune signaling in SS4, a prototype for clinically indolent, good prognosis CLL, is also relevant for the advancement of therapeutic strategies incorporating immune signaling inhibitors. Disclosures: Stamatopoulos: Roche: Research Funding.

Description: Abstract 561 Mounting evidence suggests that the molecular classification of CLL into subsets with stereotyped B cell receptors (BcRs) is functionally and clinically relevant. In fact, cases of the same subset can share not only BcR sequence motifs but also biological and clinical features as well. This suggests that the functional antigen reactivity profile of the BcR can be critical in determining the clinical features and outcome even independently of IGHV gene mutational status, at least for selected subsets. A distinctive CLL stereotyped subset, known as subset #8, is defined by the expression of unmutated, IgG-switched IGHV4-39/IGKV1(D)-39 BcRs. Subset #8 patients experience aggressive clinical courses and exhibit the highest risk for Richter′s transformation (RT) among all CLL. In order to obtain biological insight into the underlying reasons for this behavior, we profiled the antigen reactivity of subset #8 vs. other stereotyped subsets, in particular: (1) subset #1: unmutated IGHV1/5/7-IGKV1(D)-39 IgM BcRs, the largest unmutated CLL subset, with bad prognosis; (2) Subset #2: mostly borderline-mutated IGHV3-21/IGLV3-21 IgM BcRs, with bad prognosis; (3) subset #4: mutated IGHV4-34/IGKV2-30 IgG BcRs, the largest mutated CLL subset, with indolent disease. Twenty-five monoclonal antibodies (mAbs) from CLL cells were prepared as recombinant human IgG1: 11 subset #1, 6 subset #2, 3 subset #4, and 5 subset #8. The CLL mAbs were used as primary antibodies in ELISA assays against antigens which are representatives of the major classes of established antigenic targets for CLL, namely molecular structures on microbial pathogens, autoantigens and neo-epitopes created by chemical modifications during apoptosis. In particular, we tested the reactivity against lipopolysaccharides (LPS) from E. coli 055:B5, dsDNA, native BSA, malondialdehyde (MDA)-BSA, 4-hydroxynonenal (HNE)-BSA and Advanced Oxidation Protein Products (AOPP)-HSA. Subset #8 CLL mAbs exhibited broad polyreactivity as they bound to all antigens tested, showing in particular strong reactivity to BSA and MDA-BSA, E. coli LPS and dsDNA, but also against the other oxidation markers tested (HNE-BSA and AOPP-HSA), albeit to a lesser extent. This high binding was in clear contrast with the mAbs from all other stereotyped subsets. Indeed, subset #1 mAbs exhibited only medium to low reactivity against MDA-BSA and dsDNA and very low reactivity against E.coli LPS; subset #2 mAbs did not react against any of the tested antigens; and, subset #4 showed low-level reactivity only against MDA-BSA and E. coli LPS. A propos these findings, we previously demonstrated that subset #8 mAbs exhibited the strongest binding also to myosin-exposed apoptotic cells as compared to all CLL mAbs, both unmutated and mutated, including subset #1 and #2 mAbs. In addition, through in vitro functional studies of immune signaling in CLL, we observed an unrestricted and intense response of subset #8 CLL cells to ligands for Toll-like receptors (TLR) 1/2, 2/6, 7 and 9, thus differing significantly from other subsets that respond to ligands for selected TLRs only. Alltogether, these findings help to draw a scenario that may explain the particular aggressiveness of subset#8 and its increased propensity to transform. An unlimited capacity to respond to multiple immune/inflammatory stimuli present in the microenvironment may elicit unabated stimulation thoughout the natural history of these patients, leading to progressive selection of the more aggressive clonal variants. Finally, our work further indicates that immunogenetic information can be used for the rational categorization of CLL, with implications for both research and management of CLL. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: Despite the remarkable clinical results obtained with the novel kinase inhibitors i.e. the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor idelalisib in both relapsed/refractory and treatment-naïve patients, most patients achieve only partial responses underscoring the existence of resistance mechanisms that warrant further investigation. Here we explored two major mechanisms that may underlie less than optimal responses to BcR inhibition by ibrutinib, namely resistance to apoptosis due to a decreased dependence on proximal BcR signaling as it might be occurring in the context of BcR anergy; and, "bypass" activation from other non-BcR immune pathways, in particular the Toll-like receptors (TLRs). The study group included 33 CLL patients who received ibrutinib as monotherapy in 1st (n=4) or subsequent lines (n=29) of treatment. CLL cells were isolated by negative selection from peripheral blood samples collected prior to treatment initiation and, thereafter, at fixed sampling times throughout the 1st year of treatment. In keeping with the literature, we observed decreased ERK phosphorylation after 1 and 3 months of treatment as assessed by flow cytometry (p=0.0002 and

Description: Abstract 2331 Poster Board II-308 The hepatitis C virus (HCV) has been implicated in the development of B-cell lymphoproliferative disorders, including type II mixed cryoglobulinemia (MC-II) and B-cell lymphoma. MC-II is characterized by the presence of monoclonal IgM autoantibodies with rheumatoid factor (RF) activity. The monoclonal IgMs typically form immune complexes by binding polyclonal IgGs that exhibit anti-HCV reactivity. In a series of 6,196 patients affected by chronic lymphocytic leukemia (CLL), we have identified a subset of 12 cases sharing stereotyped mutated IGHV4-59/IGKV3-20 B cell receptors (BCRs) of the MD isotype (subset #13). Comparison of subset #13 heavy chain sequences to a comprehensive dataset of relevant public-database sequences revealed identical gene usage and remarkable junctional homology with the Ig sequence GenBank/U85234, the heavy chain of a RF detected in a healthy donor, as well as the sequence GenBank/AF303916, the clonotypic heavy chain from a CLL case with a history of HCV-associated MC-II. In addition, the light chain IGKV3-20/IGKJ1 stereotyped rearrangements in subset #13 were closely similar if not identical to the rearrangements expressed by clonally expanded IgM+κ+CD27+ B cells in HCV-associated MC-II. For both heavy and light chains, sequence similarities extended beyond junctional regions to shared, “stereotyped” somatic hypermutations across the entire IGHV and IGKV domain, respectively. We established viable and antibody-secreting heterohybridomas from the leukemic cells of a subset #13 case and confirmed the identity of the produced soluble antibody to the IG expressed by the CLL clone. ELISA tests against various antigens revealed that the soluble stereotyped IGHV4-59/IGKV3-20 antibody exhibited RF activity in vitro, while it was not reactive against HCV antigens. In conclusion, the present study for the first time provides evidence for the potential implication of HCV in the pathogenesis of at least a subset of CLL cases with distinctive stereotyped BCRs. The elucidation of the underlying immune mechanisms may pave the way for tailored anti-viral/anti-leukemic therapy for selected cohorts of patients that can be easily identified by molecular techniques during the diagnostic work-up. Disclosures: No relevant conflicts of interest to declare.