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Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/kappa/lambda analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/kappa/lambda thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/kappa/lambda analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10(-4)) level (n = 59) and good linearity even at the 0.001-0.01% (10(-5)-10(-4)) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach. Leukemia (2013) 27, 142-149; doi:10.1038/leu.2012.216

Introduction: The aim of this study was to screen for cell surface markers that could discriminate CLL-type MBL from CLL or identify CLL cases likely to have stable disease. Methods: Six color flow cytometry was performed on CLL-type MBL (n = 94) and CLL (n = 387) at diagnosis or relapse; 39 cases had poor-risk chromosomal abnormalities (17p and/or 11q deletion). Expression of 30 markers was analysed: CCR6, CD10, CD103, CD11c, CD138, CD200, CD22, CD23, CD24, CD25, CD27, CD31, CD38, CD39, CD43, CD49d, CD5, CD52, CD62L, CD63, CD79b, CD81, CD86, CD95, CXCR5, HLADR, IgD, IgG, IgM, LAIR1. Results: There was no difference in expression between CLL-type MBL and CLL for the majority of markers. Differential expression was observed for several markers, mainly between MBL and CLL cases with adverse-risk chromosomal abnormalities. These differences included lower expression of CD38 (9.4-fold lower, P = 0.007) and CD49d (3.2-fold lower, P = 0.008) and higher expression of LAIR-1 (3.7-fold higher, P = 0.003), CXCR5 (1.25-fold higher, P = 0.002), and CCR6 (1.9-fold higher P < 0.001) on CLL-type MBL compared to CLL with adverse chromosomal abnormalities. CD62L (L-selectin) which mediates lymphocyte adhesion to endothelial venules of lymphoid tissue, was expressed at a significantly different level between CLL-type MBL and both CLL sub-groups, with 1.3-fold lower (P = 0.04) expression levels on the MBL cases. However, there was broad overlap in expression levels. Conclusions: CLL-type MBL is phenotypically identical to CLL for a very broad range of markers. Differential expression is predominantly related to known prognostic markers and proteins involved in homing to lymphoid tissue. © 2010 International Clinical Cytometry Society

Background The C. elegans Promoterome is a powerful resource for revealing the regulatory mechanisms by which transcription is controlled pan-genomically. Transcription factors will form the core of any systems biology model of genome control and therefore the promoter activity of Promoterome inserts for C. elegans transcription factor genes was examined, in vivo, with a reporter gene approach. Results Transgenic C. elegans strains were generated for 366 transcription factor promoter/gfp reporter gene fusions. GFP distributions were determined, and then summarized with reference to developmental stage and cell type. Reliability of these data was demonstrated by comparison to previously described gene product distributions. A detailed consideration of the results for one C. elegans transcription factor gene family, the Six family, comprising ceh-32, ceh-33, ceh-34 and unc-39 illustrates the value of these analyses. The high proportion of Promoterome reporter fusions that drove GFP expression, compared to previous studies, led to the hypothesis that transcription factor genes might be involved in local gene duplication events less frequently than other genes. Comparison of transcription factor genes of C. elegans and Caenorhabditis briggsae was therefore carried out and revealed very few examples of functional gene duplication since the divergence of these species for most, but not all, transcription factor gene families. Conclusion Examining reporter expression patterns for hundreds of promoters informs, and thereby improves, interpretation of this data type. Genes encoding transcription factors involved in intrinsic developmental control processes appear acutely sensitive to changes in gene dosage through local gene duplication, on an evolutionary time scale.

e184 agents, our study shows that HBV reactivation in MM patients is not rare, and the time to reactivation differs in each case. An immunosuppressive state induced by novel agents and/or ASCT contributes to increased HBV reactivation rate. Long term monitoring of HBV DNA levels might be needed to prevent the onset of hepatitis.

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners.

A compendium of 934 transcription factor genes in C. elegans and identified by computational searches and extensive manual., Background Transcription regulatory networks are composed of interactions between transcription factors and their target genes. Whereas unicellular networks have been studied extensively, metazoan transcription regulatory networks remain largely unexplored. Caenorhabditis elegans provides a powerful model to study such metazoan networks because its genome is completely sequenced and many functional genomic tools are available. While C. elegans gene predictions have undergone continuous refinement, this is not true for the annotation of functional transcription factors. The comprehensive identification of transcription factors is essential for the systematic mapping of transcription regulatory networks because it enables the creation of physical transcription factor resources that can be used in assays to map interactions between transcription factors and their target genes. Results By computational searches and extensive manual curation, we have identified a compendium of 934 transcription factor genes (referred to as wTF2.0). We find that manual curation drastically reduces the number of both false positive and false negative transcription factor predictions. We discuss how transcription factor splice variants and dimer formation may affect the total number of functional transcription factors. In contrast to mouse transcription factor genes, we find that C. elegans transcription factor genes do not undergo significantly more splicing than other genes. This difference may contribute to differences in organism complexity. We identify candidate redundant worm transcription factor genes and orthologous worm and human transcription factor pairs. Finally, we discuss how wTF2.0 can be used together with physical transcription factor clone resources to facilitate the systematic mapping of C. elegans transcription regulatory networks. Conclusion wTF2.0 provides a starting point to decipher the transcription regulatory networks that control metazoan development and function.

Recent advances in the treatment of myeloma have included the development of immunotherapies using monoclonal antibodies targeted against plasma cell specific antigens. Elotuzumab is a therapeutic antibody directed against the SLAM family member CS1, also known as CD319, SLAMF7 or CRACC. Expression of this antigen has been investigated extensively using immunohistochemistry and gene expression profiling and has been demonstrated on normal and malignant plasma cells. Clinical trials using Elotuzumab in myeloma have shown promising results, especially in combination with other therapeutic agents, such as lenalidomide and dexamethasone. Daratumumab, a humanised antibody to CD38, has also shown encouraging responses in a percentage of refractory patients in Phase I and II trials, both as a single agent and in combination with lenalidomide. Despite this progress a significant number of patients fail to respond to these therapies for reasons which remain unclear. Monoclonal antibody-based therapy in Waldenstrom macroglobulinemia (WM) has traditionally targeted the B cell component. We have previously demonstrated that WM plasma cells are not depleted with either rituximab or alemtuzumab resulting in delayed IgM responses. Plasma cell specific antibodies may be applicable to WM and may be particularly suited to those instances when the clinical features are a consequence of the M protein such as hyperviscosity and neuropathy. There are no published data correlating quantitative surface expression data with outcome and it is possible that variability in the surface expression levels of the targets could affect efficacy of these therapeutic antibodies. The aim of this study was to evaluate the expression of CD319 and CD38 in patients with a range of plasma cell dyscrasias using multi-parametric flow cytometry. Bone marrow aspirates from patients with myeloma, MGUS or WM along with normal staging bone marrows were analysed using 8-colour flow cytometry. Leucocytes were isolated using ammonium chloride lysis and cells were then incubated with a cocktail of surface antibodies containing CD319, CD19, CD38, CD138, CD45 and CD20. Following fixation and permeabilisation cells were then incubated with Kappa and Lambda. Plasma cells and B-cells were enumerated and monoclonal B-cell and plasma cell populations were assessed. Expression of CD319 was seen on all plasma cell populations and was absent from all B-cell populations (Median fluorescent intensity (MFI) 12088 vs 114, p Although CD319 and CD38 expression was seen in all plasma cell populations, there were differences in expression levels between myeloma plasma cells and those from MGUS, WM or normal bone marrow samples. The heterogeneity in surface expression seen could potentially affect efficacy of antibody treatment and may offer some explanation for the non-responders that have been seen in early trials of Elotuzumab and Daratumumab. We have also shown that the clonal plasma cells in WM have higher levels of surface expression of both targets than those in myeloma. Following the encouraging results shown in the myeloma setting, this expression data suggests that Elotuzumab and Daratumumab may also be highly effective for eradication of the plasma cell component of WM. Prospective studies in both myeloma and WM correlating surface expression levels to outcome would be of interest. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

In past 22 years, we have identified using flow cytometry 705 patients with detectable PNH (GPI deficient) populations of granulocytes, monocytes and red cells in the peripheral blood in samples sent for diagnosis. We undertook an analysis of presenting clinical features, blood count data and PNH clone sizes in order to better understand the natural history and provide a more objective classification of disease. Based on serial flow cytometry measurements of PNH clone sizes, we also studied disease stability, frequency of recovery and progression with an aim to guiding future management of individual patients. Clinical classification of patients at presentation was as follows; aplastic anemia (58%), hemolytic anemia (36.1%); myelodysplasia (2.5%); thrombosis (2.4%); hemolysis & thrombosis (0.6%), myeloproliferative neoplasm (0.3%); Fanconi anemia (0.1%). Median age at presentation was 45 years (observed range 0.5 – 90 years) and the Male:Female ratio was 1.05. Descriptive statistical analysis of presenting blood count data revealed novel gender related features not previously described in PNH. At presentation, pancytopenia was found in 61% of male and 47% of female patients; a normal blood count was present in only 0.3% of males and 4% of females. A combined low red blood cell count (RBC) and white cell count (WBC) was the most frequent bicytopenia affecting 19% males and 22% females. Leucopenia as a sole abnormality did not occur in males and was present in5% at presentation, though this occurred in only 10/154 (6.5%) cases. For patients presenting with hemolytic disease, PNH granulocyte clones continued to increase in size in 44% of cases most likely reflecting a combination of on going selection in favour of the PNH clone and prompt diagnosis. In the 38 patients that presented with >95% granulocyte PNH clones, 92% remained stable over time (mean follow up 80 months (many on Eculizumab therapy)) with only 8% showing a gradual fall in clone size. The study shows that pancytopenia is a consistent feature of hemolytic and aplastic PNH patients. The degree of anemia is the same in both major groups of patients, but appears to be less severe for females. Not only are PNH clone sizes larger in hemolytic patients, but they also show higher platelet and leucocyte counts compared to aplastic patients, most likely reflecting a more active bone marrow. The data support the model that bone marrow failure is the primary underlying pathology in >95% of PNH patients and that sub classification on the basis of degree of aplasia, hemolysis (with or without thrombosis) and PNH clone size at presentation can be a powerful predictor of clinical course. Disclosures: Richards: Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Kelly:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

INTRODUCTION: CLL is a disorder with a wide variation in outcome. Patients with adverse cellular features are often refractory to treatment and have a short overall survival. Individuals with CLL-type MBL are unlikely to require treatment and in most cases will eventually die of an unrelated cause. Many factors that predict a poor outcome have been identified, including stage, IGHV mutation status, ZAP-70 expression, and deletions of chromosomes 17p (TP53) and/or 11q23 (ATM). Deletions and mutations in TP53 are generally not presenting features and appear to require clonal evolution. One hypothesis is that the degree of intraclonal variation in genes targeted by the somatic hypermutation machinery, e.g. IGHV and BCL6, may predict the potential for clonal evolution. We have previously tested 66 antigens for their capacity to differentiate proliferating CLL cells, resting CLL cells and normal B-cells and identified 30 potentially relevant markers, including common markers such as CD38 and less frequently used markers such as the Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). AIM: To compare the expression of relevant cell surface markers with the degree of intraclonal variation in the IGHV and BCL6 genes and to determine if these markers can be used to differentiate CLL-type MBL and CLL with or without adverse biological features. METHODS: The cell surface phenotype was assessed by 6-colour cytometry in 133 patients: 22 CLL with deletion 17p or 11q23, 69 CLL with no adverse prognostic chromosomal abnormalities, and 42 MBL. Surface phenotype was also compared with IGHV mutation status in a cohort of 29 CLL patients (16 ≤2% IGHV mutation, 13 >2% IGHV mutation). These antigens were also assessed using 4-colour flow cytometry in 20 cases (4 MBL, 16 CLL) and compared with IGHV & BCL6 mutation status and degree of intraclonal variation (defined as the proportion of mutations that were detected in a single clone only), and with ZAP-70 (AF488-1E7.2) expression. RESULTS: CLL cases with ≤2% IGHV mutation showed increased expression of CD38 (6.8 fold, p 0.02), CD49d (4.9-fold, P = 0.04), IgD (2.0-fold, P = 0.05), ZAP-70 (1.5-fold, P=0.04) and decreased expression of LAIR-1 (6.2-fold, P = 0.003) in comparison to CLL cases with >2% IGHV mutation. CLL cases with deletions of 17p and 11q23 showed decreased expression of CCR6 (1.7-fold, P = 0.0001), IgD (1.3-fold, P = 0.03) and LAIR-1 (7.1-fold, P2% overall IGHV mutation in both IGHV (median 0.075% vs. 0.049% unique mutations, P>0.05) and BCL6 (median 0.10% vs. 0.095% unique mutations, P>0.05). However, there was an inverse relationship between BCL6 and IGHV intraclonal variation and cases with the highest levels of BCL6 intraclonal variation showed significantly decreased expression of CD39 (1.9-fold, P = 0.04) and LAIR1 (4.7-fold, P = 0.019). CONCLUSIONS: There were no markers or marker combinations that could discriminate MBL from CLL. The key differences were decreased expression of markers that are expressed during cell cycle, i.e. CD23, and adhesion markers such as CD62L and CD49d. These markers show sequential changes with disease stage, supporting the hypothesis that cellular interactions are central to the accumulation and expansion of CLL cells. However, the marker most consistently associated with adverse biological features is LAIR1, which is weak or negative in CLL with ≤2% IGHV mutation, high levels of intraclonal variation and TP53 or ATM deletions. LAIR-1 is an inhibitory receptor involved in regulating classs-witching. LAIR1 is strongly expressed in normal circulating peripheral B-cells. As with other prognostic markers, expression is a continuous variable and therefore a suitable cutoff will need to be identified. However, fluorochrome-conjugated antibodies are readily available and expression on CLL cells is stable for several days in EDTA samples which should minimise inter-laboratory analytical variation. LAIR1 expression in CLL is more closely associated with IGHV mutation status than CD38 or ZAP-70 expression. LAIR1 is a promising prognostic marker that appears to be central to the development of aggressive CLL as there is a strong association between downregulation of LAIR1, intraclonal heterogeneity in BCL6 and development of TP53 and ATM deletions.

Background: Transcription regulatory networks are composed of interactions between transcription factors and their target genes. Whereas unicellular networks have been studied extensively, metazoan transcription regulatory networks remain largely unexplored. Caenorhabditis elegans provides a powerful model to study such metazoan networks because its genome is completely sequenced and many functional genomic tools are available. While C. elegans gene predictions have undergone continuous refinement, this is not true for the annotation of functional transcription factors. The comprehensive identification of transcription factors is essential for the systematic mapping of transcription regulatory networks because it enables the creation of physical transcription factor resources that can be used in assays to map interactions between transcription factors and their target genes. Results: By computational searches and extensive manual curation, we have identified a compendium of 934 transcription factor genes (r eferred to as wTF2.0). We find that manual curation drastically reduces the number of both false positive and false negative transcription factor predictions. We discuss how transcription factor splice variants and dimer formation may affect the total number of functional transcription factors. In contrast to mouse transcription factor genes, we find that C. elegans transcription factor genes do not undergo significantly more splicing than other genes. This difference may contribute to differences in organism complexity. We identify candidate redundant worm transcription factor genes and orthologous worm and human transcription factor pairs. Finally, we discuss how wTF2.0 can be used together with physical transcription factor clone resources to facilitate the systematic mapping of C. elegans transcription regulatory networks.