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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

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.

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.