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In chronic lymphocytic leukemia (CLL), increasing knowledge of the biology of the tumor cells has led to transformative improvements in our capacity to assess and treat patients. The dependence of tumor cells on surface immunoglobulin receptor signaling, survival pathways, and accessory cells within the microenvironment has led to a successful double-barreled attack with designer drugs. Studies have revealed that CLL should be classified based on the mutational status of the expressed IGHV sequences into 2 diseases, either unmutated (U) or mutated (M) CLL, each with a distinctive cellular origin, biology, epigenetics/genetics, and clinical behavior. The origin of U-CLL lies among the natural antibody repertoire, and dominance of IGHV1-69 reveals a superantigenic driver. In both U-CLL and M-CLL, a calibrated stimulation of tumor cells by self-antigens apparently generates a dynamic reiterative cycle as cells, protected from apoptosis, transit between blood and tissue sites. But there are differences in outcome, with the balance between proliferation and anergy favoring anergy in M-CLL. Responses are modulated by an array of microenvironmental interactions. Availability of T-cell help is a likely determinant of cell fate, the dependency on which varies between U-CLL and M-CLL, reflecting the different cells of origin, and affecting clinical behavior. Despite such advances, cell-escape strategies, Richter transformation, and immunosuppression remain as challenges, which only may be met by continued research into the biology of CLL.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.

Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells’ dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

Targeted therapies have been central to improvements in the treatment of hematologic malignancies. We present a review series edited by Andrew Roberts and Freda Stevenson focusing on Bruton tyrosine kinase inhibitors, phosphoinositide 3-kinase inhibitors, and BH3 mimetics and their role in the treatment of chronic lymphocytic leukemia, non-Hodgkin lymphomas and acute myeloid leukemia.

The development of follicular lymphoma (FL) from a founder B cell with an upregulation of B-cell lymphoma 2 (BCL2), via the t(14;18) translocation, to a proliferating clone, poised to undergo further transformation to an aggressive lymphoma, illustrates the opportunistic Darwinian process of tumorigenesis. Protection against apoptosis allows an innocent cell to persist and divide, with dangerous accumulation of further mutational changes, commonly involving inactivation of chromatin-modifying genes. But this is not all. FL cells reflect normal B cells in relying on expression of surface immunoglobulin. In doing so, they add another supportive mechanism by exploiting the natural process of somatic hypermutation of the IGV genes. Positive selection of motifs for addition of glycan into the antigen-binding sites of virtually all cases, and the placement of unusual mannoses in those sites, reveals a posttranslational strategy to engage the microenvironment. A bridge between mannosylated surface immunoglobulin of FL cells and macrophage-expressed dendritic cell–specific ICAM-3–grabbing nonintegrin produces a persistent low-level signal that appears essential for life in the hostile germinal center. Early-stage FL therefore requires a triad of changes: protection from apoptosis, mutations in chromatin modifiers, and an ability to interact with lectin-expressing macrophages. These changes are common and persistent. Genetic/epigenetic analysis is providing important data but investigation of the posttranslational landscape is the next challenge. We have one glimpse of its operation via the influence of added glycan on the B-cell receptor of FL. The consequential interaction with environmental lectins illustrates how posttranslational modifications can be exploited by tumor cells, and could lead to new approaches to therapy.

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

Diffuse large B-cell lymphomas (DLBCL) are a heterogeneous diagnostic entity of B-cell tumors whose behavior is variably influenced by genetic changes and environmental stimuli. They are usually divided into two major subgroups, the germinal center B-cell-like DLBCL (GCB-DLBCL) and the activated B-cell-like DLBCL (ABC-DLBCL), with different cells of origin and distinct clinical behavior. From a previous analysis of a small number of patients, we found that a fraction of DLBCL acquires N-glycosylation sites by somatic hypermutation of the tumor surface immunoglobulin (sIg) variable region, suggesting a connection with follicular lymphoma (FL). In FL, this leads to addition of mannosylated glycans in the antigen binding site (sIg-Mann), which allow interaction with microenvironmental lectins including dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN). However, the distribution and the consequences of acquired N-glycosylation motifs in DLBCL was unknown. In this study, we investigated frequency, pattern and function of the mannosylated sIg in DLBCLs. DLBCL cell lines and primary samples were analyzed for the acquisition of the N-glycosylation motifs Asparagine-X-Serine/Threonine (N-x-S/T, where x ≠ Proline) in the tumor IGHV-D-J rearranged transcripts, and for binding to DC-SIGN by flow cytometry. HILIC-UPLC and crystallography were used to define structure of the glycans located in the sIg variable region. Interaction of sIg-Mann with DC-SIGN expressing cells was measured by flow cytometry and imaged by inverted fluorescence microscopy. Intracellular signaling was measured by Phosflow. Analysis of GCB-DLBCL and ABC-DLBCL cell lines and primary samples revealed that acquired N-glycosylation sites (AGS) were common in a subset of GCB-DLBCL (51%), especially in cases with a t(14;18) translocation (88%). Remarkably, the motifs were selectively acquired in the complementary-determining-regions (CDRs) of the tumor Ig (93%). In contrast, sites were infrequent in primary ABC-DLBCL (19%) and preferentially acquired in the framework regions (51% in all ABC-DLBCL cases, 88% in IGHV4-34 ABC-DLBCL). DLBCL cell lines with AGS which bound DC-SIGN had a t(14;18) translocation and were enriched with EZH2 and KMT2D mutations, while those without AGS, and unable to bind to DC-SIGN, were not. The sites acquired in the CDRs were permissive for addition of glycans terminating at high-mannose, as revealed by immunoblotting following EndoH treatment (that digests only glycans terminating at high-mannose) of the tumor sIg and by binding to soluble DC-SIGN. This was also confirmed by HILIC-UPLC and crystal structure of a sIg-Mann+ve lymphoma-derived recombinant Fab. Binding of DC-SIGN to sIg-Mann mediated an antigen-independent signal of lower levels than that mediated by anti-Ig, as measured by increased SYK phosphorylation in the tumor B cells. The sIg-Mann+ve GCB-DLBCL, but not sIg-Mann-ve DLBCL, formed clusters round DC-SIGN expressing cells. These interactions were inhibitable or disrupted by antibodies specifically targeting the DC-SIGN carbohydrate-recognition domain. Our results refine the phenotypic and functional characteristics of a GCB-DLBCL subset, in which the cell of origin has been selected to carry glycans terminating at high-mannose in the antigen-binding region. The acquisition of sites particularly in tumors harboring the t(14;18) translocation and mutations of epigenetic modifiers suggest a cell of origin common to FL, where these features occur early at transformation. Therefore, our data suggest the presence of a tumor cell ancestor with sIg glycans and genetic features common to FL and DLBCL. These results also document that those mannoses placed in the sIg variable region are functional and engage with DC-SIGN, to receive low level signals reminiscent of those protecting B cells from apoptosis. The possibility of inhibiting this antigen-independent interaction with anti-DC-SIGN antibodies in vitro suggests a potentially exploitable way for new therapeutic intervention. Disclosures Forconi: Menarini: Consultancy; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Honoraria; Gilead Sciences: Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau.

IGHV1-69/51p1 is expressed by ∼ 30% of unmutated chronic lymphocytic leukemia (U-CLL) and combines with selected IGHD and IGHJ genes generating stereotypes if HCDR3 amino acid homology is > 60%. We had previously revealed stereotypic IGHV1-69/IGHJ6 rearrangements in normal naive B cells, thereby identifying potential counterparts of U-CLL. A different stereotypic IGHV1-69/IGHD3-16(RF2)/IGHJ3 rearrangement carrying the CAR(GGx)YD motif in the N1-region, recurrent in 6% IGHV1-69+ve CLL, is exceptionally sequence restricted, strongly suggestive of shared antigen recognition. We have now analyzed IGHV1-69/IGHJ3 rearrangements in circulating B cells of healthy individuals using several PCR-based approaches with IGHV1-69/IGHJ3 CLL sequences for reference. Stereotypes were found, but all were distinct from CLL. Remarkably, even a highly sensitive semi-nested PCR, specific for the CLL-expressed IGHV1-69/IGHD3-16(RF2)/IGHJ3 stereotype, failed to identify the CAR(GGx)YD sequence, although similar motifs were found. These highly specific B cells are not apparent in the accessible normal repertoire and may expand in response to rarely expressed antigens important in the pathogenesis of CLL.

Although long considered as a disease of failed apoptosis, it is now clear that chronic lymphocytic leukemia (CLL) cells undergo extensive cell division in vivo, especially in progressive disease. Signaling via the B-cell receptor is thought to activate proliferation and survival pathways in CLL cells and also has been linked to poor outcome. Here, we have analyzed the expression of the proto-oncoprotein MYC, an essential positive regulator of the cell cycle, after stimulation of surface IgM (sIgM). MYC expression was rapidly increased after sIgM stimulation in a subset of CLL samples. The ability of sIgM stimulation to increase MYC expression was correlated with sIgM-induced intracellular calcium fluxes. MYC induction was partially dependent on the MEK/ERK signaling pathway, and MYC and phosphorylated ERK1/2 were both expressed within proliferation centers in vivo. Although stimulation of sIgD also resulted in ERK1/2 phosphorylation, responses were relatively short lived compared with sIgM and were associated with significantly reduced MYC induction, suggesting that the kinetics of ERK1/2 activation is a critical determinant of MYC induction. Our results suggest that ERK1/2-dependent induction of MYC is likely to play an important role in antigen-induced CLL cell proliferation.

Induction of antibody-mediated immunity against hematologic malignancies requires CD4+ T-cell help, but weak tumor antigens generally fail to induce adequate T-cell responses, or to overcome tolerance. Conjugate vaccines can harness alternative help to activate responses, but memory B cells may then be exposed to leaking tumor-derived antigen without CD4+ T-cell support. We showed previously using lymphoma-derived idiotypic antigen that exposure to “helpless” antigen silences the majority of memory IgG+ B cells. Transfer experiments now indicate that silencing is permanent. In marked contrast to IgG, most coexisting IgM+ memory B cells exposed to “helpless” antigen survive. Confirmation in a hapten (NP) model allowed measurement of affinity, revealing this, rather than isotype, as the determinant of survival. IgM+ B cells had Ig variable region gene usage similar to IgG but with fewer somatic mutations. Survival of memory B cells appears variably controlled by affinity for antigen, allowing a minority of low affinity IgG+, but most IgM+, memory B cells to escape deletion in the absence of T-cell help. The latter remain, but the majority fail to undergo isotype switch. These findings could apply to other tumor antigens and are relevant for vaccination strategies aimed to induce long-term antibody.

The clue that surface Ig (sIg) is implicated in the pathogenesis of follicular lymphoma (FL) is that in FL the vast majority of the sIg variable regions are structurally modified by insertion of mannose residues into the antigen-binding sites. This is tumor-specific and reflects positive selection of sequence motifs for glycan addition introduced during somatic hypermutation. The termination at high mannoses is very unusual in cell surface molecules and it confers an ability to interact with local lectins expressed by macrophages. In this respect, the mannose cloaking of the sIg receptor resembles that of human immunodeficiency virus (HIV), which acquires a similar mannose coat to facilitate binding to macrophages. The lectin DC-SIGN is upregulated in FL, likely via local IL-4, and is the strong lectin candidate. To address the question of the functional implications of interaction between DC-SIGN and sIgM we used a FL-derived cell line (WSU-FSCCL) which expresses sIg-mannoses. We found that two recombinant derivatives of DC-SIGN (DC-SIGN-Fc or DC-SIGN-HA) bind to the sIgM but not to a cell line which express sIgM without mannose insertion. Although the mannoses are an integral part of the sIgM, and anti-IgM induces a Ca2+ flux, DC-SIGN binds but does not mimic anti-IgM. In the WSU-FSCCL cell line it remains on the surface IgM without inducing a Ca2+ response and without mediating endocytosis of the sIgM. However, DC-SIGN does act on the sIgM as revealed by the effects of pre-exposure of the cells to either DC-SIGN derivative, which, although there is no blocking of access of anti-IgM, alters the ability of the cell to respond to stimulation. Pre-exposure appears to partially paralyze the subsequent sIgM-induced Ca2+ flux indicating a lectin-mediated modification of sIgM function. On engagement by anti-IgM, sIg undergoes conventional endocytosis and this was confirmed in WSU-FSCCL. Inhibitors which block signaling did not affect endocytosis, indicating bifurcation of signaling and endocytic pathways. Knowing that DC-SIGN derivatives, again in contrast to anti-IgM, did not induce endocytosis of sIgM, we then asked if the paralysis of the sIgM-mediated Ca2+ flux by pre-exposure to lectin affected anti-IgM-induced endocytosis, and found that it did not. This confirms the separation of signaling and endocytosis and indicates that whatever change is occurring in sIgM due to lectin exposure does not remove the sIgM from the endocytic machinery. We then investigated primary FL cases, using a splenic FL and 2 lymph node FLs, all carrying N-glycosylation motifs and able to bind DC-SIGN. We focused on the DC-SIGN-HA derivative, which gives no detectable Ca2+ flux signal by itself. Binding of the lectin again paralyzed subsequent anti-IgM-induced Ca2+ flux in all cases (Fig.1A), confirming the results with the cell line. Further investigation revealed that the lectin prevents early events after antigen binding to the sIg receptor reducing SYK phosphorylation and leading to a reduced Ca2+ mobilization (Fig.1B). In conclusion, modulation of the B-cell receptor by mannose addition to the antigen-binding site allows lectin access from innate microenvironmental cells. The effect of this is to provide a low level/null signal without loss by endocytosis. The novel finding is that this interaction then lowers the function of the B-cell receptor and perhaps blocks potential interference by antigen. There may be a parallelism with reports of modification of T-cell receptor function by galectins, with both pointing to the role of post-translational modification adding another layer of control on the operation of the major immune receptors. In the case of FL, this has been exploited to maintain tumor cells in the hostile environment of the germinal center. The apparently lymphoma-specific adaptation offers opportunities for targeted inhibition of the interaction. Figure 1. Figure 1. Disclosures Forconi: Janssen-Cilag: Consultancy; Abbvie: Consultancy. Packham:Aquinox: Research Funding.

Acquisition of mannosylated glycans in the surface immunoglobulin (sIg) variable region (sIgV) antigen-binding site is a unique tumor-specific structural change of certain lymphomas, including all follicular lymphomas (FL) and ~40% diffuse large B-cell lymphomas (DLBCL). Mannosylation of the sIgV allows binding to environmental lectins including DC-SIGN (Coelho V et al, PNAS 2010). SIgV engagement is generally required for survival of DLBCL cells (Young RM et al, PNAS 2015), but how sIgV mannosylation distributes and affects behavior in the two germinal center B-cell-like (GCB-like) or activated B-cell-like (ABC-like) DLBCL subsets is unknown. While the mannosylation of the sIgV is tumor specific and irreversible, there are other natural N-glycosylation sites in the sIg constant region (sIgC). In secreted IgM these are mainly fully glycosylated and that is seen in sIgM of normal B cells (Krysov S et al, Blood 2010). However, engagement of sIgM by anti-IgM leads to expression ofan immature (mannosylated) form in both tumor and normal B cells. This conversion is dynamic, and tumor B cells restore expression of sIgC with mature glycans following BCR disengagement in vitro(Krysov S et al, Blood 2010). In this study, the glycosylation patterns of sIgV and sIgC were analyzed in GCB-like (n=6) vs ABC-like DLBCL lines (n=2) and primary samples (n=8) by IGHV-D-J sequencing, DC-SIGN binding and immunoblot of the biotinylated sIg following digestion by EndoH (specific for the mannosylated sugars) or by PNGase (removes all sugars). We found acquisition of N-mannosylation sequence motifs in the IGHV-D-J transcripts of all GCB-DLBCL lines with t(14;18), indicating a likely relationship with FL. In contrast, neither of the ABC-DLBCL lines had acquired sites, confirming a separate origin. DC-SIGN binding, which is specific for mannosylated IgV structures on the tumor cells, was observed in all GCB-DLBCL and not in the ABC-DLBCL, confirming that the acquired sites were glycosylated. These results allowed us to discriminate DLBCL cases into "DC-SIGN binders" (DB-DLBCL) vs "DC-SIGN non-binders" DLBCL (NB-DLBCL). Analysis of the carbohydrate structures on the sIgC revealed that the immature form was confined to the NB-DLBCL lines (2/2), while the DB-DLBCL expressed a mature fully glycosylated form (6/6). Consistent with the nature of ABC-DLBCL, these results revealed an activated BCR status of the NB-DLBCL. This was confirmed in the 8 primary samples (5/8 DB, 3/8 NB), which expressed an immature (activated) sIgC in 3/3 NB-DLBCL and a mature sIgC in 5/5 DB-DLBCL. However, engagement of anti-IgM F(ab')2 polyclonal antibody converted the inactive sIg form of DB-DLBCL into an activated sIg with relative increase of the immature sugars. It was evident that the mannosylated sites on the sIgC were not available for DC-SIGN binding, which is confined to the sIgV sites. We verified BCR activation status by investigating constitutive phosphorylation of SYK, BTK and PLCγ2, which are recruited to the membrane upon BCR activation, prior to endosome formation (Phelan JD et al, Nature 2018), in 2 DB-DLBCL lines (NU-DHL1 and SU-DHL6) and 2 NB-DLBCL (HBL-1 and TMD8). Basal phosphorylation of SYK, BTK and PLCγ2 was higher in the NB-DLBCL, consistent with the activated status associated with an immature sIgC. Our results reveal a functional dichotomy in DLBCL, which indicates: first, the cell of origin dictates whether sIgV carries mannoses in the antigen-binding site; second, reversible sIgC mannosylation associates with activation via sIg. Interestingly, this feature of activation is in ABC-DLBCL, which lacks IgV mannosylation. It is consistent with the suggestion that occupation of the antigen-binding sites with mannoses blocks further engagement of the receptor by 'antigen'. However, acquisition of mannoses in the sIgV sites appears to confer an ability to interact with environmental lectins such as DC-SIGN, whereas the sIgC sites fail to do this, suggesting an alternative function. Clearly, the post-translational modification targets several sites in sIg. Sites in the sIgC have a similar, possibly maturational, function in normal B cells, but in tumor cells the irreversible addition of mannoses to the sIgV adds a tumor-specific function. Disclosures Packham: Aquinox: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.

Introduction: Follicular lymphoma (FL) cells retain expression of a functional B cell receptor (BCR) despite the loss of one Ig allele due to the hallmark t14:18 translocation and ongoing somatic hypermutation (SHM) of the variable genes (V genes) which increases the likelihood of crippling mutations. SHM introduces N-glycosylation (N-gly) motifs within the V genes, a feature exclusively restricted to germinal centre (GC)-derived lymphomas. Oligosaccharides of the high mannose type are added to motifs and interact with calcium-dependent lectins associated with cells of the microenvironment. This activates the BCR signalling pathway and likely contributes to the survival, retention and proliferation of tumor cells in the GC. Determining at what stage of disease evolution N-gly motifs are acquired and their behaviour during progression can ascertain their importance in pathogenesis and their potential as an effective therapeutic target. To achieve this, we analysed motifs within the Ig heavy chain variable gene (IGHV) of tumor-related subclones across temporal FL samples. Method: Genomic DNA from three FL patients taken at different time points of disease progression were analysed. In total, 8 samples were selected, all carrying an IGHV3 rearranged tumor clone. IGHV DNA amplicons were sent for 2x250bp paired-end sequencing using the Miseq Illumina platform (Genewiz, NJ). Tumor-related reads with counts greater than ten were selected following analysis on IMGT/HIGH-V-QUEST. Reads were aligned and unique sequences were assigned as subclones. Additional tumor related reads sequenced on the Roche 454 Life Sciences Genome Sequencer FLX were available (Patients 4 & 5). Subclones were analysed for N-gly motifs and evolutionary pathways were generated using the IgTree program, based on intraclonal SHM profiles and homology of tumor clones to the germline IGHV sequence. Results: The earliest time point samples for Patient's 1, 3, 4 & 5 contained one N-gly site within the IGHV of the MC defined by the largest count number. These sites were conserved in >97% of unique subclones (p97% of subclones in and across disease events, whereas the FR3 site was conserved in 95.5% of the diagnostic subclones and in ~80% of the relapsed and transformed populations. No subclones with loss of all four sites were detected for Patient 2. Patient 5 samples were taken from different anatomical sites with tumor populations acquiring distinct N-gly motifs, suggesting an early divergence in tumor evolution. Despite this, a minor population of motif positive clones are shared, suggesting a trafficking ability of subclones. Subclones with motifs made up ≥99% of the total tumor count, highlighting the motif as a feature of the tumor bulk, with motif negative clones representing a minor population. These negative clones are presumably lost during disease progression as they not shared between events. Evolutionary analysis revealed no additional sites are gained as motif positive clones expand while rare negative subclones cannot reacquire sites and do not undergo further diversification. Conclusion: We report for the first time that acquired N-gly motif sites are a clonal feature in FL disease as seen through their conservation both in the heterogeneous subclonal population and the overall tumor mass. The sites are also retained in progression-associated subclones while rare motif-negative subclones disappear. This suggests that although acquisition of additional driver mutations may dampen the tumor's microenvironment dependency, the motifs and added mannoses may retain functional significance at later stages of disease. The data indicates motifs as being a universal event of the reservoir cell pool responsible for propagating disease episodes. Targeting N-gly sites and their interacting partners may lead to the disruption of an early and vital FL-microenvironment interaction, presumably mediated through the mannose-lectin interaction, reducing relapse rates and progression of disease. Disclosures Forconi: Abbvie: Consultancy; Janssen-Cilag: Consultancy. Gribben:Abbvie: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Cancer Research UK: Research Funding; Wellcome Trust: Research Funding; Acerta Pharma: Honoraria, Research Funding; TG Therapeutics: Honoraria; NIH: Research Funding; Kite: Honoraria; Janssen: Honoraria, Research Funding; Unum: Equity Ownership; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Lymphoid tumors have been pivotal in connecting cell biology to clinical observation, and this continues. The deluge of genomic and epigenomic information is increasing our understanding of pathogenesis and progression. However, in contrast to the known hallmark chromosomal changes, for example

Ken H. Young, MD, PhD, shares some highlights of this review series. The success of antibodies in fighting infection is clear, but directing this power against cancer cells has been a challenge. For cancer, antibodies are usually made externally and injected into patients, where they act against

The cell of origin of chronic lymphocytic leukemia (CLL) has long been sought, and immunoglobulin gene analysis provides new clues. In the unmutated subset (U-CLL), there is increased usage of the 51p1-related alleles of the immunoglobulin heavy chain variable 1-69 gene, often combined with selected genes and with immunoglobulin heavy chain diversity IGHJ6. Stereotypic characteristics of the HCDR3 result and suggest antigen selection of the leukemic clones. We have now analyzed 51p1/IGHJ6 combinations in normal blood B cells from 3 healthy persons for parallel sequence patterns. A high proportion (33.3% of sequences) revealed stereotypic patterns, with several (15.0%) being similar to those described in U-CLL. Previously unreported CLL-associated stereotypes were detected in 4.8%. Stereotypes (13.6%) not detected in CLL also were found. The HCDR2-IGHJ6 sequences were essentially unmutated. Junctional amino acids in normal B cells were heterogeneous, as in cases of stereotyped CLL. Phenotypically, normal B cells expressing 51p1-derived immunoglobulin M were naive. This snapshot of the naive B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded immunoglobulin M of normal B cells suggest a restricted sequence repertoire shaped by evolution to recognize common pathogens. Proliferative pressure on these cells is the likely route to U-CLL.

Primary central nervous system (CNS) lymphoma is an aggressive B-cell tumor that is defined clinically by the absence of systemic disease. We have used immunoglobulin variable (V)–gene analysis to identify tumor cells at the CNS site in 12 cases and to probe the involvement of peripheral tissues in 3 patients. Clonal tracking revealed tumor cells in the bone marrow and/or blood for 3 of 3 cases, with evidence for increased V-gene mutational activity at peripheral sites. In 2 of 3 cases, intraclonal variant analysis revealed identity with the brain biopsy but detected additional variants unique to extracerebral sites. These findings suggest that peripheral tumor cells can undergo separate development locally with no reentry into the brain. Primary CNS lymphoma appears to have both CNS-specific and systemic components with limited interchange. The more malignant behavior of tumor cells in the CNS suggests either a local environmental influence or a less malignant phenotype of the peripheral clone.

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I–binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2–restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT137–45–specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT137–45, have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

Although the presence or absence of somatic mutations in the immunoglobulin variable region (IgVH) genes in chronic lymphocytic leukemia (B-CLL) identifies subtypes with very different prognoses, the assay is technically complex and unavailable to most laboratories. CD38 expression has been suggested as a surrogate marker for the 2 subtypes. IgVHmutations and CD38 expression in 145 patients with B-CLL with a long follow-up were compared. The 2 assays gave discordant results in 41 patients (28.3%). Multivariate analysis demonstrated that Binet stage,IgVH mutations and CD38 were independent prognostic indicators. Median survival time in patients whose cells had unmutated IgVH genes and expressed CD38 was 8 years; in those with mutated IgVHgenes not expressing CD38, it was 26 years. For those with discordant results, median survival time was 15 years. Thus, although CD38 expression does not identify the same 2 subsets as IgVHmutations in CLL, it is an independent risk factor that can be used with IgVH mutations and clinical stage to select patients with B-CLL with the worst prognoses. Using cryopreserved cells taken at intervals during the course of the disease, however, changes of CD38 expression over time were demonstrated in 10 of 41 patients. Causes of the variation of CD38 expression require further study. Additional prospective studies are required for comparing CD38 expression with other prognostic factors and for taking sequential measurements during the course of the disease.

Current treatment strategies for chronic lymphocytic leukemia (CLL) involve a combination of conventional chemotherapeutics, monoclonal antibodies, and targeted signaling inhibitors. However, CLL remains largely incurable, with drug resistance and treatment relapse a common occurrence, leading to the search for novel treatments. Mechanistic target of rapamycin (mTOR)-specific inhibitors have been previously assessed but their efficacy is limited due to a positive feedback loop via mTOR complex 2 (mTORC2), resulting in activation of prosurvival signaling. In this study, we show that the dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor PF-04691502 does not induce an mTORC2 positive feedback loop similar to other PI3K inhibitors but does induce substantial antitumor effects. PF-04691502 significantly reduced survival coincident with the induction of Noxa and Puma, independently of immunoglobulin heavy chain variable region mutational status, CD38, and ZAP-70 expression. PF-04691502 inhibited both anti-immunoglobulin M-induced signaling and overcame stroma-induced survival signals and migratory stimuli from CXCL12. Equivalent in vitro activity was seen in the Eμ-TCL1 murine model of CLL. In vivo, PF-04691502 treatment of tumor-bearing animals resulted in a transient lymphocytosis, followed by a clear reduction in tumor in the blood, bone marrow, spleen, and lymph nodes. These data indicate that PF-04691502 or other dual PI3K/mTOR inhibitors in development may prove efficacious for the treatment of CLL, increasing our armamentarium to successfully manage this disease.

The molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) has been highlighted by gene expression profiling (GEP), dividing DLBCL into the following three main molecular subtypes with different clinical outcomes and responses to immunochemotherapy: the germinal center B-cell-like (GCB) subtype, the activated B-cell-like (ABC) subtype and the primary mediastinal B-cell lymphoma (PMBL) subtype. Despite frequent translocations involving the IGH (heavy chain) locus, B-cell receptor (BCR) expression is retained in almost all DLBCL, indicating that BCR signaling is crucial for the survival of malignant cells. Furthermore, DLBCL are characterized by recurrent somatic mutations that are supposed to be oncogenic drivers. Here, with the aim to more accurately define the DLBCL pathogenic subgroups, we report a detailed immunogenetic analysis of IGH rearrangements in a well-defined GEP DLBCL cohort and correlate these features with the somatic mutation status of recurrently mutated genes involved in lymphomagenesis. Methods: 204 DLBCL enrolled in the prospective LYSA LNH03 trial program were classified into molecular subtypes by GEP using Affymetrix arrays [82 ABC (40.2%), 77 GCB (37.7%), 29 (14.2%) unclassified and 16 PMBL (7.8%)]. The amplification of complete VDJ rearrangements was realized using BIOMED-2 protocols. Somatic hypermutation (SHM) characteristics were studied (mutation rate, glycosylation N-X-S/T sites, RGYW hotspots, composition of CDR3) and correlated with the molecular subtypes. To obtain a more comprehensive molecular portrait of the DLBCL cases, GEP and IGH VDJ analyses were correlated with the mutational status of a panel of 34 recurrently mutated genes in DLBCL such as MYD88, EZH2, CD79B, TNFAIP3, CARD11 and GNA13 (Dubois et al. Clinical Cancer Research 2016). Results: A total of 153/204 (75%) clonal VDJ rearrangements were successfully determined. Failures to detect clonotypic sequences were predominantly seen in PMBL (56%) and unclassified (31%) cases. The ABC subtype display a significantly lower SHM rate than the 3 other subtypes, especially in contrast to the PMBL subgroup that was characterized by a higher SHM rate (p Disclosures Haioun: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: 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. Salles:Mundipharma: Honoraria; Janssen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding.

Introduction: mRNA translation is increased in activated tumor cells of the aggressive form of Chronic Lymphocytic Leukemia (CLL), typically unmutated (U) immunoglobulin gene heavy-chain variable region (IGHV) with a strong sIgM signaling capacity (Yeomans et al, Blood 2016). C-MYC protein is a master regulator of cell performance and its expression is controlled at both transcriptional and translational levels. C-MYC protein is over-expressed in the proliferation centers of CLL and high c-MYC mRNA expression is associated with poor prognosis. In leukemic cell lines, c-MYC is an essential mediator and direct target of NOTCH1. Pro-activating c.7541_7542delCTmutations in NOTCH1 PEST domain of chromosome 9 exon 34 (NOTCH1ΔCT) are enriched in U-CLL with high sIgM levels/signaling capacity and associate with poorer prognosis in CLL (D'Avola et al, Blood, 2016), likely due to accumulation of more stable NOTCH1 protein and enhanced signaling in tissue activated CLL cells (Arruga et al, Leukemia, 2014). Aims and Methods: We investigated the consequences of NOTCH1ΔCT on global mRNA and c-MYC translation using a novel flow cytometry-based O-propargyl-puromycin (OPP) incorporation assay ('Click-iT' assay) and by c-MYC-specific immunoblotting in U-CLL. Since prolonged culture of CLL cells in vitro in the absence of stimuli led to spontaneous inactivation of NOTCH1 pathway, CpG-mediated TLR9 induction was used as a tool for activation of CLL cells in vitro. Cycloheximide (CHX) was used as a negative control for mRNA translation. For this study, 2 cohorts were investigated: i) a test "CLLΔCT cohort" of U-CLL with NOTCH1ΔCT (variant allele frequency [VAF] by droplet digital PCR, range 42.6-48.9%, median 47% of the CD19+CD5+ CLL cell population), but no additional genetic lesion other than 13q deletion, and ii) a control "CLLWT cohort" of U-CLL with no NOTCH1ΔCT (VAF Disclosures Steele: Portola Pharmaceuticals: Honoraria. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding.

In chronic lymphocytic leukemia (CLL), B-cell receptor (BCR) signaling is key to survival and proliferation, and can be effectively targeted by the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. BCR surface IgM (sIgM) levels and signaling capacity are low but variable in CLL. The variability has clinical consequences and relatively high sIgM in circulating cells is associated with aggressive disease, a higher proliferative component and higher levels of adhesion and migration receptors (D'Avola, Drennan et al, Blood 2016). The aim of this study was to investigate the expression and function of sIgM and other surface markers on the circulating tumor cells of CLL patients following one week of ibrutinib therapy. Expression of markers associated with the BCR complex (IgM, IgD, CD19), and with adhesion or migration (CD38, CD49d, CXCR4) was assessed by flow cytometry in 13 CLL patients prior to and following one week of therapy. Signaling capacity was measured by immunoblotting following sIgM or CXCR4 stimulation. N-glycosylation status of the µ-heavy chains was measured by immunoblotting after biotinylation of cell surface proteins to determine IgM maturation (Krysov et al, Blood 2010). Following one week of treatment basal activity of BTK was significantly reduced compared with pre-treatment (P=0.002), and anti-IgM induced BTK auto-phosphorylation was completely suppressed, confirming full inhibition of BTK in vivo. Expression of sIgM, but not of sIgD, increased dramatically (P=0.005), with little change in the other receptors. The increase was similar in the tissue-egressed CXCR4dimCD5hi cells and in the CXCR4brightCD5low long-resting cells of the circulation. Increased sIgM expression associated with the fully N-glycosylated form of sIgM, characteristic of the absence of antigen engagement. Also, it correlated with increased capacity to phosphorylate SYK following anti-IgM stimulation (r=0.91, P Whilst ibrutinib induces a rapid and persistent redistribution of CLL cells into the blood, early suspension is typically associated with reduction of the lymphocytosis, nodal progression and short survival. These data reveal a dangerous early phenomenon during ibrutinib in which IgM expression has increased in the circulating cells, resulting in enhanced IgM-mediated signaling capacity through SYK. CXCR4 inhibition may prevent migration into tissue during ibrutinib and avoid (super)antigen encounter, but cells could be at risk of increased IgM mediated proliferation and disease progression if ibrutinib is withdrawn at this stage. Disclosures Packham: Aquinox Pharmaceuticals: Research Funding; Karus Therapeutics: Other: Share Holder & Founder. Steele:Portola Pharmaceuticals: Honoraria.

Signaling via the B-cell receptor (BCR) is a major driver of malignant B-cell proliferation/survival in chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma. The role of kinases in BCR signaling is well understood and kinase inhibitors are effective therapies for these diseases. However, resistance is increasingly common and new drugs are required. SHIP1 is a PI(3,4,5)P3-specific phosphatase which suppresses PI(3,4,5)P3-dependent signaling downstream of PI3 kinase (PI3K) and imposes "inhibitory" activity via accumulation of its product PI(4,5)P2. Here, we investigated the effects of AXQ-C5, a novel chemical activator of SHIP1, on BCR signaling and expression of CXCR4, a chemokine receptor which is thought to play an important role in tissue homing and/or retention of CLL cells in vivo. Immunoblot analysis revealed that SHIP1 was constitutively tyrosine phosphorylated in malignant cells derived from the blood of chronic lymphocytic leukemia (CLL) patients. BCR stimulation (with anti-IgM) did not substantially alter SHIP1 expression or phosphorylation, but resulted in rapid (within 30 minutes) relocalization of phospho-SHIP1 to the plasma membrane. Confocal imaging demonstrated that a fraction of this plasma membrane-associated phospho-SHIP1 co-localized with surface IgM (sIgM). Induced association of sIgM and phospho-SHIP1 following anti-IgM treatment was confirmed by co-immunoprecipitation. Pre-treatment of CLL cells with AQX-C5 resulted in significant reduction in the levels of anti-IgM-induced phosphorylation of ERK1/2 and AKT. AQX-C5 also significantly reduced induction of the MYC oncoprotein and, in longer time-course experiments, induced caspase-dependent CLL cell apoptosis. By contrast, T cells were relatively resistant to the pro-apoptotic effects of AQX-C5. AQX-C5 also induced apoptosis in diffuse large B-cell lymphoma cell lines dependent on chronic BCR signalling. We also investigated the effects of AQX-C5 on expression of CXCR4. Interestingly, AQX-C5 alone was sufficient to cause a strong down-modulation of CXCR4 expression (~50%) in the majority of samples analyzed. This effect was relatively specific since other cell surface receptors, including the transferrin receptor (which like CXCR4 is subject to rapid endocytosis), were unaffected by AQX-C5. Interestingly, idelalisib did not result in CXCR4 down-modulation, suggesting response to AQX-C5 is a consequence of PI(4,5)P2 accumulation rather than decreased levels of PI(3,4,5)P3 per se. Moreover, AQX-C5 was much less effective in inducing CXCR4 down-modulation in normal B cells derived from healthy donors, suggesting this response to AQX-C5 may be leukemia-specific. In conclusion, this study supports the hypothesis that chemical SHIP1 activation is sufficient to suppress BCR-mediated signaling, acting, at least in part, by decreasing PI(3,4,5)P3-mediated activation of the AKT pathway. In addition, SHIP1 activation results in strong CXCR4 down-modulation, a response not observed with direct PI3Kdelta inhibition or in normal B cells. Combined inhibitory effects on BCR signaling and CXCR4 expression make SHIP1 activators exciting new therapeutic agents for B-cell cancers, potentially including those that have acquired resistance to kinase inhibitors. Disclosures Packham: Aquinox Pharmaceuticals: Research Funding; Karus Therapeutics: Other: Share Holder & Founder. Steele:Portola Pharmaceuticals: Honoraria. Cragg:Bioinvent International: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Baxalta: Consultancy; Gilead Sciences: Research Funding; GSK: Research Funding. Cross:Aquinox Pharmaceuticals (Canada), Inc.: Employment, Equity Ownership. Harwig:Aquinox Pharmaceuticals (Canada), Inc.: Employment, Equity Ownership. Mackenzie:Aquinox Pharmaceuticals (Canada), Inc.: Employment, Equity Ownership.

B-cell receptor (BCR) mediated signalling is pivotal to the pathogenesis of chronic lymphocytic leukaemia (CLL), which is broadly split into two major subsets based on the immunoglobulin heavy chain variable (IGHV) gene mutational status. Progressive disease is characterised by unmutated IGHV genes (U-CLL) and more indolent disease by mutated IGHV genes (M-CLL). Antigen/autoantigen engagement is crucial for BCR-driven CLL cell survival, disease progression and resistance to therapy. Drugs that target this pathway are revolutionising the treatment of this disease. However, these inhibitors are not curative and some patients develop resistance to ibrutinib following mutation of the BTK or PLCγ2 genes or for as yet unknown reasons. We have shown that IL-4 pre-treatment induced sIgM expression especially in cases with unmutated IGHV genes, augmented anti-IgM induced signalling and compromised the ability of ibrutinib (1 µM) and idelalisib (1 µM) to inhibit BCR-mediated signalling. Previous reports highlighted a role of miR-155 (Cui Blood 2014) and miR150 (Mraz Blood 2014) in regulating BCR signalling in CLL cells by targeting SHIP1 (negative regulator of BCR signalling) and GAB1 and FOXP1 (positive regulators of BCR signalling) respectively. In this study we investigated the effect of IL-4 on expression of these miRNA and demonstrated that IL-4 augments and inhibits expression of miR155 and miR150 respectively (Figure 1), but had no effect on the house keeping control gene RNU6, demonstrating a mechanism whereby IL-4 augments BCR signalling. Furthermore, since BCR engagement can promote adhesion in CLL cells, we investigated the effect of IL-4 alone and in combination with anti-IgM on integrin expression such as CD49d, CD49e and CD29 and on adhesion. IL-4 pre-treatment augmented CD49d (α4) and CD44 expression and promoted anti-IgM as well as PMA mediated adhesion of CD5\CD19+ CLL cells to fibronectin. Interestingly in contrast to greater sIgM increases by IL-4 in U-CLL, IL-4 promoted greater anti-IgM-induced adhesion largely in the M-CLL subset. This indicates that IL-4 may have distinct effects in the two subsets. Given our results, we hypothesised that dual inhibition of IL-4 and BCR signalling may promote greater responses in patients. At ASH 2015 we demonstrated that cerdulatinib, a dual JAK/SYK inhibitor, now in phase II clinical trials in CLL, can inhibit IL-4 and anti-IgM/anti-IgD induced signalling at concentrations achievable in patients (Cmin 1μM). We have now extended these studies and shown that cerdulatinib treatment prevented IL-4-induced effects such as increased sIgM expression and subsequent downstream signalling, and prevented downmodulation of CXCR4 and to a greater extent than the specific JAK3 inhibitor tofacitinib. Furthermore, inhibition of JAK1/3 by cerdulatinib also reduced IL-6, IL-10, IL-15, IL-21 and IFNγ-induced signalling, which have all been shown to play a role in CLL biology. More importantly, cerdulatinib killed CLL cells under basal conditions, particularly in cases with progressive disease. However, CLL cells within the patient lymph nodes likely receive signals from the BCR, IL-4 and possibly CD40L, which results in the elevation of pro-survival proteins Mcl-1 and Bcl-XL, providing protection from basal and ibrutinib or venetoclax induced killing. Following simultaneous treatment with bead-immobilised anti-IgM, 10ng/ml IL-4 and 300ng/ml CD40L, cerdulatinib was still able to induce a modest amount of apoptosis, however in combination with venetoclax there was a synergistic increase in killing. Together these data provide evidence of how IL-4 increases BCR signalling by directly modulating miRNA expression, but also additional effects on adhesion which may subsequently retain the tumour cells within the lymph nodes. JAK inhibition may prevent these effects. Therefore, these findings indicate that JAK inhibition in combination with BCR kinase inhibition maybe of value for the treatment of CLL. Figure. Q-PCR analysis of miR-155 and mIR-150 expression following treatment with or without IL-4 for 24h. Figure. Q-PCR analysis of miR-155 and mIR-150 expression following treatment with or without IL-4 for 24h. Disclosures Conley: Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Davies:Bayer: Research Funding; Karyopharma: Honoraria, Research Funding; GSK: Research Funding; Janssen: Honoraria; Pfizer: Research Funding; Mundipharma: Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel to scientific conferences, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodation, expenses, Research Funding. Johnson:Celldex Therapeutics: Research Funding. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Coffey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Steele:Portola Pharmaceuticals: Honoraria.

Primary diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors accounting for approximately 40% of B-cell malignancies. The immunoglobulin (Ig) variable region genes have undergone rearrangement and are commonly somatically mutated. The majority show intraclonal variation which indicates that somatic mutation has continued after transformation. Typically, cells of DLBCLs express Ig of a single isotype, but there may be accompanying cells that express alternative isotypes. To probe the status of the isotype switch process in DLBCL, 4 cases of tumor-derived constant region transcripts of all isotypes were investigated. Following the identification of the VDJ sequences, the presence of the major isotype expected from immunohistochemical analysis was confirmed at the RNA level. Another 3-4 alternative isotypes were revealed in all cases, some of which could also be detected by immunohistochemistry. All cases were somatically mutated with an intraclonal variation. In 2 cases there were clearly distinct patterns of somatic mutation between isotypes, which was consistent with independent evolution of the tumor subpopulations. There was apparent clustering of mutational patterns into either an IgMD/IgG3/IgA set or an IgG1/IgA set, indicating that the switch to IgA can occur by different routes. Alternative isotype expression is evident in DLBCL at both the RNA and protein levels. The pattern of mutation indicates that switching is occurring in subpopulations of the tumor after malignant transformation. The findings support the concept that isotype switch events may be a feature of DLBCL.

Despite having several characteristics of naı̈ve B cells, chronic lymphocytic leukemia (CLL) cells have been shown in some cases to have somatically mutated Ig variable region genes, indicating that the cell of origin has passed through the germinal center. A previous study of patients with CLL found an association between lack of somatic mutation and trisomy 12 and, therefore, possibly with a less favorable prognosis. We have sequenced the Ig VH genes of the tumor cells of 84 patients with CLL and correlated our findings with clinical features. A total of 38 cases (45.2%) showed ≥ 98% sequence homology with the nearest germline VH gene; 46 cases (54.8%) showed >2% somatic mutation. Unmutated VH genes were significantly associated with V1-69 and D3-3 usage, with atypical morphology; isolated trisomy 12, advanced stage and progressive disease. Survival was significantly worse for patients with unmutated VH genes irrespective of stage. Median survival for stage A patients with unmutated VH genes was 95 months compared with 293 months for patients whose tumors had mutated VHgenes (P = .0008). The simplest explanation is that CLL comprises 2 different diseases with different clinical courses. One, arising from a memory B cell, has a benign course, the other, arising from a naı̈ve B cell, is more malignant.

Despite having several characteristics of naı̈ve B cells, chronic lymphocytic leukemia (CLL) cells have been shown in some cases to have somatically mutated Ig variable region genes, indicating that the cell of origin has passed through the germinal center. A previous study of patients with CLL found an association between lack of somatic mutation and trisomy 12 and, therefore, possibly with a less favorable prognosis. We have sequenced the Ig VH genes of the tumor cells of 84 patients with CLL and correlated our findings with clinical features. A total of 38 cases (45.2%) showed ≥ 98% sequence homology with the nearest germline VH gene; 46 cases (54.8%) showed >2% somatic mutation. Unmutated VH genes were significantly associated with V1-69 and D3-3 usage, with atypical morphology; isolated trisomy 12, advanced stage and progressive disease. Survival was significantly worse for patients with unmutated VH genes irrespective of stage. Median survival for stage A patients with unmutated VH genes was 95 months compared with 293 months for patients whose tumors had mutated VHgenes (P = .0008). The simplest explanation is that CLL comprises 2 different diseases with different clinical courses. One, arising from a memory B cell, has a benign course, the other, arising from a naı̈ve B cell, is more malignant.

Primary central nervous system lymphoma (PCNSL) represents 1% to 3% intracranial tumors. Most PCNSL are located in the brain, and 75% are large B-cell lymphomas. The largest subgroup of these tumors contains cells that resemble centroblasts and has been labelled diffuse centroblastic (polymorphous) lymphoma. To investigate the cell of origin and the clonal history of these tumors, we have analyzed VH gene of 5 cases of PCNSL, all confirmed by histological studies to be Epstein-Barr virus (EBV)-negative, high-grade diffuse B-cell lymphomas. The V4-34 gene of the VH4 family was used in 4 of 5 cases. All VHgenes were found to have accumulated very high levels of somatic mutation (14% to 25%). In 3 of 5 cases, intraclonal nucleotide heterogeneity, including codon deletion in some clones in 1 case, was observed, indicating that the VH genes were still under the influence of the somatic hypermutation mechanism. Analysis of the distribution of silent and replacement mutations showed evidence for preservation of immunoglobulin structure in all cases. These results suggest that, although there is no evidence for germinal center formation in the brain tissue, PCNSL is derived from a B cell with features associated with location in a germinal center environment.

Investigations of VH gene mutational patterns in B-cell tumors are often performed at an arbitrary time point of disease. To assess the effects of disease progression, tumor-derived VHgenes have been monitored from presentation through treatment and relapse in one patient with follicle center lymphoma (FCL), and two patients with primary diffuse large B-cell lymphoma (DLCL). The patient with FCL and one patient with DLCL both achieved clinical remission, although this was only partial in the FCL. However, both subsequently relapsed, and the second patient with DLCL was refractory to radiotherapy and chemotherapy. In each case, the tumor-derived VH sequence was identified, and the CDR3 “clonal signature” was used to track tumor cell sequences in subsequent biopsies. All cases showed somatic mutations, with intraclonal heterogeneity evident at presentation, and some sequences were aberrant. The VH sequences of the DLCL which responded to treatment became homogeneous at relapse. The sequences of both the FCL and the refractory DLCL remained heterogeneous. In all cases, transcripts of multiple Ig isotypes could be identified, and there was immunophenotypic evidence for expression of several Ig isotypes. The case of refractory DLCL had identifiable transcripts from IgM, IgD, IgA, IgG, and IgE, but appeared to lose the ability to produce alternative isotype transcripts and protein at the late stage of disease. These cases indicate that VH gene analysis can be used to probe tumor cell behavior in cases of lymphoma and that perturbations caused by therapy and disease progression can occur.

An unusual group of human B-cell tumors with cellular features of chronic lymphocytic leukemia or lymphoplasmacytoid leukemia, together with high levels of a monoclonal IgG serum protein, has been investigated. Analysis of tumor-derived VH genes of neoplastic B lymphocytes was used to determine the clonal relationship between the IgM expressed or secreted by the tumor cells and the IgG serum paraprotein. In all five cases, VH gene sequences showed transcripts of IgM and IgG of common clonal origin. Sequences were derived from VH3 (4 of 5) and VH1 (1 of 5) families and were all highly somatically mutated with strong evidence for antigen selection. There was no intraclonal variation detectable in either IgM or IgG sequences. In 3 of 5 cases, in which monoclonal IgM and IgG were found in serum, the VH genes combined to Cμ or Cγ showed identical mutational patterns. However, in 2 of 5 cases, in which IgM was confined to cell expression with only monoclonal IgG in serum, sequences of the VH transcripts of IgM and IgG showed many shared mutations but also numerous differences. In these cases, the level of mutation was similar in IgM and IgG and both appeared to be antigen selected. In summary, the final neoplastic event in this group of tumors has apparently occurred at the point of isotype switch from IgM to IgG, leading to dual isotype synthesis. In the group that secreted both isotypes, the mutation pattern was identical, indicating either synthesis by a single cell, or silencing of mutational activity before switching. In the group that did not secrete IgM, cells of each isotype were distinct and reflected a divergent mutational history.

Chronic lymphocytic leukemia (CLL) is a B-cell tumor involving small lymphocytes that generally express the CD5 antigen and low levels of surface Ig. Within this definition, there is heterogeneity among cases in cell morphology, karyotypic abnormalities, and clinical course. Trisomy 12, the most frequent karyotypic abnormality, is commonly found in a subset of CLL with atypical morphology. It has also been associated with advanced disease, and possibly with a less favorable prognosis. A further subset of cases with abnormalities involving chromosome 13q14 have typical lymphocyte morphology. Occasionally, the two abnormalities are found together. To assess the clonal history of the cell of origin in disease subsets defined by these two chromosomal abnormalities, we investigated the usage of VH genes and the pattern of somatic mutation in 10 cases of trisomy 12 with atypical morphology and eight cases of 13q14 abnormality with typical morphology. In addition, four cases with both chromosomal abnormalities were analyzed. Results confirm a common usage of the VH1 family in all subsets. However, the patterns of somatic mutation were distinct, with cases of trisomy 12 showing a minimal level of mutation (mean ± SD, 0.34% ± 0.86%) and cases of 13q14 abnormality showing significant levels (6.5% ± 1.67%). The four cases with both abnormalities showed a mixed pattern. All mutated cases had intraclonal homogeneity, and three of 10 had a pattern indicative of antigen selection. These results suggest that the clonal history of the two subsets of CLL may differ.

In multiple myeloma, sequence studies of VH genes used to encode clonal Ig in neoplastic plasma cells have shown a common pattern of extensive somatic hypermutation. A further consistent feature of these VH sequences is a complete lack of intraclonal variation. These findings indicate that the malignant cell arises at a mature, postfollicular stage of B-cell development. However, only a minority of cases have a distribution of somatic mutations in VH consistent with a prior role for antigen in selecting the B cell of origin. To complement these studies, and to take further the investigation of a role for antigen in the clonal history of myeloma, we have investigated tumor-derived VL sequences from bone marrows of 15 patients. All sequences (9Vκ and 6Vλ) were potentially functional and 5 of 15 had evidence for N-region additions. All had undergone extensive somatic hypermutation, and showed no intraclonal variation. In 4 of 15 cases, the distribution of mutations revealed a significant (P < .05) clustering of replacement mutations in the CDR sequences, indicating a role for VL in selection by antigen. Comparison with the VH sequences used by the same tumor cells showed that, if significant clustering was present, it was in either VH or VL, but not both. Altogether, 10 of 15 V-regions showed evidence for antigen selection, suggesting that the B cell of origin has behaved as a normal germinal center B cell. Deductions concerning a role for antigen selection may require both VH and VL sequences for validation.

Tumor cell lines and one tumor biopsy from seven cases of Epstein-Barr virus (EBV) genome-negative sporadic Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig variable region genes. The VH genes were derived from the VH 3 (one) and VH4 (six) families and both the IgM-positive (six) and the IgA-positive (one) were all mutated from their germline counterparts. The VL genes were derived from V kappa 1 (one), V kappa 3 (one), V lambda 1 (four), and V lambda 2 (one) families and were also somatically hypermutated. Biopsy material from one of the IgM-positive cases showed VH and VL sequences that matched the derived cell line, with additional intraclonal sequence heterogeneity, indicating that the tumor cells had undergone posttranformation somatic mutation. Mutational patterns in V(H) genes did not show a conventional role for antigen in selecting tumor cell sequences. In contrast, patterns in VL sequences were consistent with a role for antigen in five of seven cases. The pattern of extensive scattered somatic hypermutation and intraclonal variation is similar to that in VH sequences of EBV genome-positive endemic BL, although the degree of mutational activity is less. These common features indicate that B cells involved in the two variants of BL may share a common clonal history.

Plasma cell tumors display a wide spectrum of clinical progression, ranging from aggressive multiple myeloma to a benign form known as monoclonal gammopathy of undetermined significance (MGUS), which requires no treatment. Because both diseases involve mature Ig- secreting plasma cells, the reason for this variation in malignant behavior is unclear. However, assessment of malignant potential is desirable for choice of treatment protocols. Ig variable (VH) gene sequences analysis has previously shown the tumor cell of multiple myeloma to be postfollicular, with mutated homogeneous clonal sequences indicating no continuing exposure to the somatic hypermutation mechanism, and this was confirmed in 7 of 7 patients. Comparison of the VH gene sequences in the monoclonal cells in MGUS yielded a different result, with 3 of 7 patients demonstrating mutated heterogeneous sequences consistent with the tumor cells remaining under the influence of the mutator. In 1 of 3 of these patients, an IgM-positive precursor cell was identified that expressed heterogeneous VH sequences similar to those of the isotype-switched plasma cell. These results indicate that the clonal cells in MGUS differ from those in myeloma and suggest that the difference may reflect malignant potential.

Tumor cell lines from six typical cases of endemic Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig VH genes. The neoplastic cells all had a t(8;14) (q24;q32) translocation involving the c-myc protooncogene. The VH genes were derived from VH1, VH3 and VH4, and both the IgM-positive (four cases) and IgG-positive (two cases) were extensively mutated from germline sequence. In two cases, early and late passage tumor cells were available, and the VH nucleotide sequences were identical, indicating that mutations had not accumulated in vitro. In a further case, there was evidence of sequence heterogeneity, which appeared to have been generated in vivo, indicating that the tumor cell VH gene was able to undergo posttranslocation somatic hypermutation. Analysis of the relatively nonpolymorphic VH4 genes for the pattern of replacement or silent mutations did not show a role for antigen selection in the expressed sequences.

Splenic lymphoma with villous lymphocytes (SLVL) is a recently defined subgroup of chronic B-cell lymphoproliferative diseases. The characteristic morphology of the tumor cells, together with phenotypic and cytogenetic findings, indicate that it is a distinct entity, but the nature of the cell or origin and its relationship to other low- grade lymphomas is unclear. For B-cell tumors, analysis of the variable region heavy chain (VH) genes used to encode the clonal Ig has shown marked differences between histologic categories, both in gene usage and extent of somatic mutation. An investigation of VH genes used in five typical cases of SLVL has shown somatic hypermutation from germline sequences in all cases, indicating that the cell of origin has been exposed to the hypermutation mechanism. However, no clonal heterogeneity was detectable, demonstrating that the tumor cell does not accumulate further mutations. These characteristics are similar to those found in mature postfollicular B cells, such as plasma cells. The distribution of mutations leading to replacement amino acids differed among the cases, with three of five cases showing clear evidence for antigen selection.

Monoclonal gammopathy of undetermined significance (MGUS) can transform to multiple myeloma (MM). In myeloma, mutated VHgenes with sequence homogeneity reveal a postfollicular origin. Previously, some MGUS cases showed mutated VH genes with intraclonal variation, indicating an earlier stage of arrest. We investigated progression from 2 of 2 MGUS to MM, in which VH genes confirmed clonal evolution. In one MGUS case, intraclonal heterogeneity was evident, and transformation to myeloma occurred rapidly with apparent homogeneity in the emergent clone. However, residual MGUS-derived sequences were detectable at this time. Heterogeneity in MGUS does not associate with benign disease, but it indicates an origin from a tumorigenic cell, most likely surface immunoglobulin+, undergoing somatic mutation. The remaining case displayed intraclonal homogeneity at the MGUS stage, conceivably resulting from a self-cloning outgrowth from MGUS with heterogeneity. Transformation can occur at either MGUS stage, but it involves a single cell in which somatic mutation is then silent.

Follicular lymphoma (FL) is a B-cell tumor arising in germinal centers and retaining features of its normal B-cell counterpart. Lymphomagenesis appears stepwise from the t(14;18) translocation, through FL-like cells, to FL in situ, then to overt FL. Surface Ig is mandatory and carries a striking V-region modification because of introduction of glycan addition sites during somatic mutation. These are positively selected and acquire unusual high mannoses, which interact with lectins. The Ig-associated mannoses appear essential for FL, providing a disease- specific target for antibody attack. Antibody therapy is currently focused on anti-CD20 (rituximab), which appears to rely predominantly on the Fcγ module recruiting suitably activated macrophages. Immunogloblulin and, to some extent, CD20, can each escape antibody attack in vitro by modulation, but this is difficult to demonstrate clinically. Instead, studies of anti-CD20 therapy of FL suggest that effector modulation, similar to that seen in the suppression of autoimmune inflammation by infusions of normal human IgG, may be important. Both antigenic and effector modulations might be minimized by repeated small doses of more potent antibodies. Clearly, mechanisms of attack vary with the malignancy, the target molecule, and the antibody design, offering opportunities for optimizing this promising strategy.

The B-cell receptor (BCR) of chronic lymphocytic leukemia (CLL) cells with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable (IGHV) regions shows a variable degree of anergy, associated with low surface IgM (sIgM) levels and signaling capacity, more evident in M-CLL. DNA methylation is also different between M-CLL and U-CLL. Patients from both subsets are immunosuppressed from the early stages, possibly influenced by the ability of activated CLL cells to produce IL-10, a property of B10 cells. The aim of this study was to investigate associations between capacity of circulating CLL cells to differentiate into B10 cells and features of anergy. In parallel, the methylation status of the IL-10 gene locus was probed in order to investigate associations between IL-10 gene methylation and production. CLL cells were isolated and cultured +/- TLR9 activation with CpG (ODN-2006). Cytokine secretion was measured in supernatants (Luminex) and IL10 transcript was measured (RT-qPCR) from cell pellets. IL-10 production by CLL cells was measured by intracellular flow cytometry following stimulation with CpG for 24 hours. Epigenetic profiling was performed using MassARRAY and the 450K Array. We found that activation of CLL cells by CpG consistently induced production and secretion of IL-10 protein, while no or low amounts of pro-inflammatory cytokines were detected. Production of IL-10 was significantly higher in M-CLL than in U-CLL. Levels also correlated with lower sIgM levels and signaling capacity, both features of anergy. A linear correlation was present between secretion, intracellular production and transcript levels of IL-10, suggesting no aberrant post-transcriptional controls. In the absence of CpG activation, IL-10 transcript levels were also detected at low levels in M-CLL, while they were even lower in U-CLL. To identify a potential basis for the differential expression, DNA methylation analysis of IL-10 gene locus was performed. While the promoter region displayed similarly low levels of methylation in both U-CLL and M-CLL, methylation differences were detected immediately downstream of the promoter within the first intron (approximately +200 to +500 bp, differentially methylated region 1, DMR1) and in the gene body (approximately +1300 to +1800 bp, DMR2). Both DMR1 and DMR2 were markedly more hypomethylated in M-CLL than U-CLL, with the greatest difference detected at cg17067005 within DMR1. Each of these regions display histone H3 lysine 27 acetylation (H3K27ac) in B cells (GM12878 cells), thus are likely to represent functional DNA elements. Analysis of IL-10 transcript levels with methylation demonstrated a mutually exclusive pattern between expression and methylation of both DMR1 and DMR2. These data document a strong link between capacity to differentiate into B10-like cells and anergy, and suggest an epigenetic component in the regulation of IL-10 production in CLL cells. This capacity may contribute to immunosuppression. While U-CLL appears less able to produce IL-10 on a per cell basis, higher tumor load may compensate, accounting for clinical immunosuppression in both subsets. Disclosures No relevant conflicts of interest to declare.

Chronic lymphocytic leukaemia (CLL) is characterised by an accumulation of B cells which is broadly split into two groups representing a progressive IGHV unmutated (U-CLL) and a more indolent IGHV mutated (M-CLL) disease. Activation of the B cell receptor (BCR) by antigen/autoantigen engagement is crucial for CLL cell survival, disease progression and resistance to therapy, however further research is required to better understand how BCR signalling impacts on CLL biology. Autophagy is known to play a role in tumorigenesis and resistance to therapy in solid tumors, however whether autophagy has a role in CLL biology and how it is regulated has not been fully investigated. Autophagy is important for normal B cell development and is known to be regulated by various drug treatments in vitro in CLL samples. A previous study showed that activation of the BCR on murine splenic B cells with soluble or bead immobilised (BI) anti-IgM induced autophagy and subsequent apoptosis, however, the role of BCR-induced autophagy has not been explored in B cell malignancies and particularly CLL. Firstly, we assessed basal protein expression of key autophagy markers LC3BII, and ATG3 in CLL samples and age-matched normal donor B cells (NDB). CLL cells expressed significantly more LC3BII (p=.014, n=57) and ATG3 (p=.04, n=58) compared with NDB (n=8), with a greater LC3BII protein expression in U-CLL compared to M-CLL (p=.039, n=57), indicating more autophagy occurs in U-CLL. Furthermore basal increases in autophagy markers GABARAPL2 (LC3B family member) (p=.0004, n=34) and ATG4A (p=.04, n=20) at the RNA level were significantly associated with the ability of CLL cells to flux calcium (>10%) in response to anti-IgM. This indicated a possible role of the BCR in the regulation of autophagy in CLL samples and a possible association with progressive disease. Activation of the CLL BCR with BI anti-IgM significantly induced expression of autophagy markers ATG3 (p=.002, n=22), LC3BII (p Disclosures Strefford: Roche: Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.

The emergence of B cell receptor (BCR) kinase inhibitors has proved effective for the treatment of a number of B-cell malignancies including chronic lymphocytic leukemia (CLL). BTK and PI3K inhibitors have clear efficacy in suppressing tumor progression but have not been curative. A number of patients have developed resistance to these drugs following mutation of the BTK or PLCγ2 gene. Whilst, other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. Thus the development of novel drugs which are still effective once other BCR-kinases inhibitors become ineffective is of paramount importance. Spleen tyrosine kinase (Syk) is essential for B cell receptor signalling pathways as well as a variety of other surface receptors such as MHCII, FC receptors and integrins, all of which have been shown to play a role in CLL biology. Importantly, Syk inhibition has been shown to overcome resistance to ibrutinib, identifying Syk inhibition as a promising strategy to treat these patients. Furthermore, we have previously shown that IL-4 is found in CLL lymph nodes and can promote resistance to ibrutinib and idelalisib by restoring αIgM induced calcium flux and phosphorylated ERK (ASH 2014, abstract #3299). IL-4 signalling is mediated through the JAK/STAT signalling pathways via JAK1 and JAK3, therefore simultaneous inhibition of both Syk and JAK1/3 may be therapeutically beneficial over BCR kinase inhibitors alone. Cerdulatinib (PRT062070) is a dual JAK/Syk inhibitor in a phase I open label dose escalation study and is currently demonstrating clinical activity in patients with relapsed/refractory B cell malignancies including CLL. Our group has now demonstrated in vitro that cerdulatinib, at plasma concentrations achievable in patients, can induce apoptosis of CLL cells in a concentration and time dependent manner with a mean IC50 of 3µM and 1µM at 48 and 72h respectively, defined by annexin V/PI and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Apoptosis was caspase dependent since treatment with the pan caspase inhibitor ZVAD.fmk significantly inhibited cerdulatinib induced cell death at 24h. Cerdulatinib induced apoptosis coincided with an increase in pro-apoptotic proteins Noxa and Puma and a decrease in the anti-apoptotic protein Mcl-1. Cerdulatinib significantly inhibited IL-4 induced phosphorylation of STAT6 at 300nM (p=.005), BCR induced phosphorylation of AKTS473 with soluble (p=.008) and bead immobilised (BI) (p=.025) αIgM at 30nM and phosphorylation of AKTT308 with BI αIgM at 300nM (p=.008). Furthermore, in patients with CLL, it is thought that CD40L and IL-4 are key factors, which promote survival of CLL cells in proliferation centres within the lymph node microenvironment. Therefore, we cultured CLL cells with a vehicle control or IL-4\CD40L, prior to treatment with cerdulatinib. Cerdulatinib alone induced similar levels of apoptosis irrespective of IL-4/CD40L treatment, suggesting cerdulatinib may be able to overcome microenvironmental signals and target cells within the lymph node. Next we explored the possibility of augmenting cerdulatinib induced apoptosis by simultaneous inhibition with the Bcl-2\Bcl-XL inhibitor ABT-199. In vitro in the presence of IL-4/CD40L, ABT-199 synergised with cerdulatinib to induce significantly greater cell death than with either agent alone. Therefore these data provide in vitro evidence for the use of cerdulatinib in clinical trials for the treatment of CLL as either a single agent or in combination with other therapies such as ABT-199. Disclosures Strefford: Roche: Research Funding. Davies:Seattle Genetics: Research Funding; Takeda: Honoraria. Coffey:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.

Idiotypic determinants on neoplastic B cells could provide tumor antigens for vaccination of patients with B-cell tumors. Because this approach requires an individual vaccine for each patient, simple methods for obtaining idiotypic antigen are desirable. Using polymerase chain reaction (PCR) with family-based V-gene and J-region primers, the variable region genes of heavy and light chains (VH and VL) of Ig have been obtained from biopsy material from 13 patients with B-cell tumors. In each case, analysis of random clones derived from the PCR product showed repeated, clonally-related sequences, whereas normal lymphoid tissue generated no repeated sequences. In 3/3 cases, the repeated sequences were found to be the same as those in a tumor-derived hybridoma. Mutational patterns in the V-genes differed among the tumors, with follicular lymphoma tending to be more highly mutated. The individual VH and VL sequences have been assembled with a flexible linker sequence to encode single-chain Fv (scFv). The scFv sequences can be cloned into bacterial expression vectors to produce protein, or into vectors suitable for direct vaccination using naked DNA. In a model system, expressed scFv protein retained all idiotypic determinants defined by a panel of five anti-idiotypic monoclonal antibodies (MoAbs). Similarly, expressed scFv proteins from two patients were shown to react with anti-idiotypic antibodies. This approach allows production of potential vaccines from surgical biopsies within 2 to 3 weeks.

B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIMEL and BIML, in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIMEL only. In CLL, anti-IgM–induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIMEL serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIMEL/BIML phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIMEL phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.

The B-cell receptor (BCR) is a key survival molecule for normal B cells and for most B-cell malignancies. Recombinatorial and mutational patterns in the clonal immunoglobulin (Ig) of chronic lymphocytic leukemia (CLL) have revealed 2 major IgMD-expressing subsets and an isotype-switched variant, each developing from distinct B-cell populations. Tracking of conserved stereotypic features of Ig variable regions characteristic of U-CLL indicate circulating naive B cells as the likely cells of origin. In CLL, engagement of the BCR by antigen occurs in vivo, leading to down-regulated expression and to an unanticipated modulation of glycosylation of surface IgM, visible in blood cells, especially in U-CLL. Modulated glycoforms of sIgM are signal competent and could bind to environmental lectins. U-CLL cases express more sIgM and have increased signal competence, linking differential signaling responses to clinical behavior. Mapping of BCR signaling pathways identifies targets for blockade, aimed to deprive CLL cells of survival and proliferative signals. New inhibitors of BCR signaling appear to have clinical activity. In this Perspective, we discuss the functional significance of the BCR in CLL, and we describe strategies to target BCR signaling as an emerging therapeutic approach.

A monoclonal anti-idiotypic antibody has been raised that recognizes Igs with heavy chains encoded by a member of the VH4 family, the VH4–21 gene segment. The idiotope (Id) is detectable on a high percentage of early B cells in fetal spleen, and is expressed by certain autoantibodies, particularly cold-reactive anti-red blood cell antibodies. Therefore, it was of interest to investigate usage of this VH gene by neoplastic B cells; 81 chronic lymphocytic leukemias (CLLs) involving CD5+ B cells and 62 B-cell lymphomas of varying histologic type have been analyzed. The Id was expressed by only 3 of 81 (3.7%) of the CLLs, indicating a relatively low usage by these tumors. In contrast, the Id was expressed by 9 of 62 (14.5%) of the lymphomas across a range of histologic types, indicating a differential use of the VH4–21 gene among B-cell neoplasms. For three of the Id-positive lymphomas, each of a different histologic class, the nucleotide sequence of the tumor-derived VH gene was determined; the VH4–21 gene was identified, as expected. The sequence from the CLL was identical to the germline sequence, and the marginal zone lymphoma showed only 3 nucleotide changes, 2 of which gave rise to amino acid substitutions. In contrast, the sequence from the follicular lymphoma showed 29 nucleotide changes giving rise to 14 amino acid substitutions, which were scattered among the CDR and FW regions.

Surface IgM (sIgM) has a key influence on the clinical behavior of chronic lymphocytic leukemia (CLL). We now report that it exists in 2 forms with different N-glycosylation patterns in the μ-constant region. One glycoform is similar to normal B cells in bearing mature complex glycans common to most cell-surface glycoproteins. The other is an immature mannosylated form more characteristic of μ chains in the endoplasmic reticulum. Unmutated CLL (U-CLL) expresses a higher proportion of mannosylated surface μ chains than mutated CLL. Normal B cells express only the mature glycoform but can express the immature form after persistent engagement of sIgM, suggesting that glycan modification is a consequence of antigen exposure. CLL cells express variable proportions of the mannosylated form and can revert to the mature form after incubation in vitro. Both glycoforms are able to signal after sIgM engagement in vitro, leading to enhanced tyrosine phosphorylation. These findings support the concept that CLL cells are continuously exposed to antigen in vivo, driving the N-glycosylation pattern of expressed sIgM toward a mannosylated form, especially in U-CLL. Strikingly, this is reminiscent of follicular lymphoma, where mannosylated Ig is expressed constitutively via N-glycosylation sites in the variable region, suggesting a functional asset for this glycoform.

To the editor: Immunoglobulin V genes and CD38 expression in CLL The finding that the mutational status of immunoglobulin (Ig) V genes correlates with identifiable disease subsets of chronic lymphocytic leukemia (CLL) and is of prognostic importance has been documented and mutually corroborated by