ALBERT

Description: Introduction Multiple myeloma (MM) is a largely incurable plasma cell malignancy characterised by marked genomic heterogeneity, in which chromosome 1q21 amplification (amp1q21) associates with poor prognosis. Genomic analysis using next generation sequencing has identified recurrent mutations, but no universal acquired somatic mutation(s) have emerged in MM, suggesting that understanding pathways of survival will require analysis of individual tumours in distinct disease subsets. To compound complexity of the problem, intraclonal variation (ICV), known as a major driver mechanism in cancer plasticity, in which clonal competitor cells undergo selection during disease evolution and progression by Darwinian principles, will need to be fully mapped at the genome level. Identifying the true level of ICV in a tumour will thus require analysis at the level of whole exome sequencing (WES) in single cells (SCs). In this study, we sought to establish WES methodology able to identify ICV in SCs in an index case of amp1q21 MM. Methods Cell selection and sequencing CD138+ tumour cells and CD3+ T-cells were isolated from a presentation case of amp1q21 MM as bulk populations to high purity (〉97%). Single MM cells and normal T cells were individually isolated and used for single cell (SC) whole exome sequencing (WES). Whole genome amplification (WGA) was performed by multiple displacement amplification (Qiagen REPLI-g Mini kit), and exome capture was performed using Agilent SureSelect. Libraries were then 90 bp paired end sequenced on an Illumina HiSeq2000 (BGI, China). Data analysis Data was produced for bulk (1000 cells) MM and bulk germline T cells, twenty MM SCs and five T cell SCs. Raw data was aligned to hg19 reference sequence using NovoAlignMPI (v3.02.03). Variant calling was performed using SAMtools (v1.2.1) and VarScan (v2.3.6) and variants were annotated using ANNOVAR. High confidence variants were called in the bulk tumour WES by pairwise comparison with bulk germline WES. Variant lists were also cross-searched against various variant databases (CG46, 1000 genomes, dbSNP, esp650 and in-house database) in order to exclude variants that occur in the general population. Multiple quality control measures were employed to reduce the number of false positive calls. Results and Discussion Data and bioinformatics pipelines are of a high quality SC WES generated raw data reads that were similar to bulk WES of 1000 cells, with comparable mapping to Agilent SureSelect target exome (69-76% SC vs. 70% bulk) and mean fold coverage (56.8-59.1x vs. 59.7x bulk). On average, 82% of the exome was covered sufficiently for somatic variant (SV) calling (often considered as ≥ 5x), which was higher than seminal published SC WES studies (70-80%) (Hou et al., Cell, 2012; Xu et al., Cell, 2012). We identified 33 potentially deleterious SVs in the bulk tumour exome with high confidence bioinformatics, 21 of which were also identified in one or more SC exomes. The variants identified include suspected deleterious mutations in genes involved in MAPK pathway, plasma cell differentiation, and those with known roles in B cell malignancies. To confirm SV calls, randomly selected variants were validated by conventional Sanger sequencing, and of 15/15 variants in the bulk WES and of 55/55 variants in SCs, to obtain 100% concordance. Intraclonal variation in MM Significantly, ICV was apparent from the SC exome variant data. Total variant counts varied considerably among SCs and most variant positions had at least several cells where no evidence of the variant existed. Bulk WES lacks crucial information We identified an additional 23 variants that were present in 2+ SC exomes, but absent in the bulk MM tumour exomes. Of these, 30% (7 variants) were examined for validation, and were amplifiable in at least one cell to deliver 100% concordance with variant calls. These variants are of significant interest as they reveal a marked occurrence of subclonal mutations in the MM tumour population that are not identified by bulk exome sequencing. They indicate that the mutational status of the MM genome may be substantially underestimated by analysis at the bulk tumour population level. Conclusion In this work we establish the feasibility of SC WES as a method for defining intraclonal genetic variation in MM. Disclosures No relevant conflicts of interest to declare.

Description: A functional BCR is essential for survival of normal B-cells, in response to cognate antigen or tonic, antigen-independent stimuli. Cross-linking of BCR by antigen triggers key phosphorylation events in the early signalosome. Mammalian wild type BRAF, a member of the RAF cytosolic kinase family (A-RAF, B-RAF, C-RAF) is critical to BCR function, mediating RAS-RAF-MEK-ERK or mitogen-activated protein kinase (MAPK) pathway signal transduction, via phosphorylation of ERK1/2. The MAPK pathway is a central conduit for survival and proliferation. Importantly, use of inducible deletion models have shown that wild type BRAF is the predominant kinase that transduces BCR signals to activate ERK1/2, with C-RAF playing an accessory role and A-RAF displaying a x20-30 fold lower basal activity in ERK1/2 stimulation. Tonic BCR survival signals in normal B-cells are also transduced via phosphorylation of ERK1/2, but do not require antigen stimuli. Whether the BCR plays a comparable role in sustaining survival in malignant B-cells is less well characterized and remains a central question in understanding the origins and progression of mature B-cell neoplasms. Hairy cell leukaemia (HCL) is a rare B cell leukaemia with characteristic hair-like cytoplasmic projections on tumor cells, displaying distinctive activation markers. A striking feature of monoclonal HCL tumors is expression of multiple variant immunoglobulin heavy chain (sIgH) isotypes as components of the BCR on individual tumor cells, defining the major subset of disease (mult-HCL). Most mult-HCL tumors exhibit IGV somatic hypermutation with a low level of on-going mutations and AID expressed, implicating initial contact with antigen via the BCR. The functional relevance of the BCR to transformation and survival however has as yet not been mapped in HCL. Seminal exome sequencing data in typical HCL identified mutant BRAF V(600)E as almost universal in this tumor. Consistent with mutant BRAFV(600)E, levels of phosphorylated ERK (p-ERK) are raised in hairy cells. As there is an essential requirement for wild type BRAF in transducing BCR signals, the question arises how mutant BRAF may affect BCR function in HCL, given requirements for dimerization of wild type BRAF for ERK1/2 activation, and whether ERK1/2 activation can be enhanced by functional BCR signals for downstream effector signals in HCL. To examine this, we evaluated BCR signalling in mult-HCL (n=10), in cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ mult-HCL, IgD mediated persistent Ca2+ flux, which was also evident via 〉1 sIgH isotype, linked to increased ERK1/2 activation and BCR endocytosis (4/4 cases). In sIgD-vemult-HCL however (6/6 cases), BCR-mediated signals activating ERK1/2 for downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal a clear discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing mult-HCL (3/3 cases), only a single sIgL was fully functional. We next examined effects of anti-BCR stimuli on mult-HCL tumor cell survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both sets (4/4 cases) to establish a consistent outcome in these replicate cases. IgD stimuli, in marked contrast retained tumor viability with no evidence for pre-apoptotic effects (2 cases; 1 mult-HCL and 1 sIgD only-HCL). Despite mutant BRAF, BCR signals amplify ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL lacking IgD, a role for antigen in stimulating the BCR for tumor persistence appears unlikely. It remains feasible that mutant BRAF in these cases retains tonic signals through activated ERK1/2 for survival, but this remains speculative at present. Surface IgD emerges with potential to transduce BCR signals for tumor survival and persistence in-vivo, but given its enigmatic role even in normal B-cells, its relevance to HCL progression is unclear. These observations raise the possibility that mutant BRAF may be a mechanism to bypass any antigen-dependent BCR signalling constraints in mult-HCL. Disclosures: No relevant conflicts of interest to declare.

Description: 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.

Description: Abstract 1567 The B-cell receptor (BCR) is critical to survival of normal B-cells, and regulates key aspects of cellular behavior. Of these, response to antigen determines pathways of normal B-cell maturation, including isotype switch events that occur by deletional class switch recombination (CSR), an irrevocable event, to yield IgG/A memory B-cells. Less frequently, CSR via a cryptic site generates IgD+ B-cells whereas IgM+IgD+ antigen experienced B-cells synthesize each isotype by an alternative transcript splicing mechanism. The role of the BCR in survival of malignant B-cells however is less well defined, in particular in response to antigen. Intriguingly, in Hairy cell leukemia (HCL), BCR assembly occurs with multiple surface immunoglobulin (sIg) isotypes (mult-HCL), many co-expressed on individual hairy cells (HCs) in an otherwise monoclonal tumor. Multiple isotypes appear to exclude deletional CSR events, and suggests a RNA processing mechanism of molecular assembly. This phenotype is rare even amongst malignant B-cells, and raises the question of the functional relevance of individual variant isotypes. It also potentially presents a model to dissect roles of multiple isotypes on single B-cells. To examine this, we investigated the BCR in CD19+CD11c+CD103+ mult-HCL cases (n=10), in which 2–4 differing sIg isotypes were present on most HCs, with single or, in 3 cases, dual sIgL expression. In all cases, IGHV genes were mutated, and confirmed monoclonality. Phenotype revealed 2 distinct subsets by sIg isotype co-expression, IgD+ve and IgD-ve. Using Ca2+ flux and ERK phosphorylation assays after cross-linking with specific anti-sIg antibodies, we observed a functional BCR in all mult-HCL examined, in both subsets (10/10 cases Ca2+, 6/6 cases ERK). However, striking differences emerged between the two subsets. In sIgD+ve mult-HCL, IgD mediated persistent Ca2+ flux, with flux also evident via 〉1 sIgH isotype. In marked contrast, in sIgD-ve mult-HCL Ca2+ flux was restricted to a single sIgH isotype, but not via IgM. Flux signals in this subset were transient. In most cases only a single sIgL transduced flux. We next evaluated BCR endocytosis after cross-linking individual isotypes and IgL. In 2 sIgD+ve cases, anti-IgD and anti-Igλ stimulation led to endocytosis of both sIgD and sIgλ, and in 1 case, where examined, anti-IgM stimulation endocytosed both sIgM and sIgλ. In 3 sIgD-ve cases, functional sIgH and sIgL induced endocytosis of the stimulated isotype, but again sIgM was dysfunctional, remaining immobilized on the cell surface. Ca2+ flux through endocytosed isotypes was correspondingly either significantly reduced or ablated in both subsets. In HCs, BCR endocytosis is clearly dependent on functional isotypes and IgL, and parallels events in normal B-cells. Lastly, we examined downstream effects of BCR signalling on cell viability, using soluble (sAb) and bound (bAb) anti-sIg antibodies. In a single IgD+ve mult-HCL case, both sAb and bAb anti-IgM yielded a significant level of apoptosis compared to control antibodies, whereas anti-IgD sAb resulted in no appreciable difference to level of spontaneous apoptosis, suggesting a disengagement of signals from this pathway. This disengagement was also observed in a separate HCL case expressing only IgD, and not in the mult-HCL cohort initially selected, where anti-IgD signals again did not increase levels of apoptosis. In IgD-ve mult-HCL (n=4), sAb and bAb specific cross-linking of IgG/A triggered significant apoptosis. These data demonstrate, for the first time, that mult-HCL retains a functional responsiveness via the BCR, suggesting an absence of anergic effects that may follow chronic antigen exposure in-vivo to self-antigen. Signals via sIgM/G/A isotypes, where functional, induce apoptosis in mult-HCL, whereas sIgD opposes such effects. Despite an apparently unique molecular mechanism of IgD expression in mult-HCL, this isotype appears to be hardwired in B-cells to mediate responses that differ from IgM. The persistent flux observed here indicates a more sustained and robust IgD signaling cascade, as also observed in B-cell models. These data reveal distinctive and opposing effects of individual isotypes on BCR mediated behavior in mult-HCL. While apoptotic responses appear to negate a role for antigen in tumor drive in-vivo, potential antigen engagement via IgD, if dominant leaves this question open. Disclosures: No relevant conflicts of interest to declare.

Description: Somatic hypermutation (SHM) and isotype class switch recombination are critical mechanisms that diversify normal B-cells in response to antigen, and are generally confined to the germinal center (GC). Post-GC B-cells differentiate either into memory B-cells or antibody forming cells maturing as plasma cells. Memory B-cells which exit the GC have been regarded as invariably expressing CD27 and this population comprises both IgM+ and switched B-cells. It is, however, the presence of SHM that remains the defining feature of memory status in B-cells. Recently, it has been reported that dye extruding membrane ATP-binding transporters expressed by naive B-cells can distinguish them from memory B-cells which lack transporters and this, notably, led to the identification of IgG+ memory B-cells that did not express CD27. However, these observations were not correlated with SHM status. Here, we have examined these findings further and focused specifically on IgM+D+ B-cells in relation to our interest in mapping origins of B-cell tumors. Using dye-based transporter assays, we identified a substantial IgM+CD27− population in PBMNC from healthy individuals which lacked transporter activity. These cells were next isolated using a different strategy, employing IgD and CD27 expression, and a highly purified (〉99%) IgD+CD27− fraction obtained by FACS sorting. There was virtually nil contamination by CD27+ B-cells. IgM transcripts were then specifically analyzed in this CD27− population for VH gene mutations by using Cmu downstream primers. We examined VH3 and VH5 gene transcripts from 2 separate sorts from a healthy donor following amplification by primary and nested RT-PCR and cloning products for sequence analysis. Of 104 VH3 clones from this CD27− fraction, 38 (37%) were germline and the remainder mutated (55% with 98–99.9% homology to germline, 9%