The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

MRD assessment by MFC was predictive of overall outcome in patients with myeloma undergoing ASCT. This predictive value was seen in patients achieving conventional CR as well as patients with favorable and adverse cytogenetics. The effects of maintenance strategies can also be evaluated, and our data suggest that maintenance thalidomide can eradicate MRD in some patients.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Key Points• The level of MRD quantified by flow cytometry is more informative than a 0.01% threshold and independently predicts OS.• There was approximately 1 year survival benefit per log depletion. A lower cut point for predicting improved outcome was not reached.The detection of minimal residual disease (MRD) in myeloma using a 0.01% threshold ( 10 24 ) after treatment is an independent predictor of progression-free survival (PFS), but not always of overall survival (OS). However, MRD level is a continuous variable, and the predictive value of the depth of tumor depletion was evaluated in 397 patients treated intensively in the Medical Research Council Myeloma IX study. There was a significant improvement in OS for each log depletion in MRD level (median OS was 1 year for ‡10%, 4 years for 1% to <10%, 5.9 years for 0.1% to <1%, 6.8 years for 0.01% to <0.1%, and more than 7.5 years for <0.01% MRD). MRD level as a continuous variable determined by flow cytometry independently predicts both PFS and OS, with approximately 1 year median OS benefit per log depletion. The trial was registered at www.isrctn.com as #68454111. (Blood. 2015;125(12):1932-1935

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/κ/λ 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/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ 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.

Somatic mutations at methionine 41 (Met41) in UBA1, encoding the major E1 enzyme responsible for initiating ubiquitylation, were recently identified as the cause of a novel autoinflammatory disease, named VEXAS (Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). We sought to determine the prevalence of UBA1 mutations in a UK cohort of patients matching the VEXAS clinical phenotype. We identified 10 new patients with somatic mutations in UBA1, but only 8 had altered p.Met41. A novel variant, c.167C&gt;T; p.Ser56Phe was identified, which was present in myeloid, and not lymphoid lineages and led to preferential loss of the catalytic activity of cytoplasmic UBA1. An additional novel variant, c.118-1G&gt;C was identified at the splice acceptor site of exon 3 leading to altered splicing in vitro. Bone marrow biopsies from two patients with a Met41 substitution and the novel splice site variant were consistent with previously reported features of VEXAS. The bone marrow of the patient with the p.Ser56Phe variant was less similar, likely driven by a distinct but overlapping disease mechanism. Our study therefore confirms somatic p.Met41 substitutions in UBA1 as a major cause of VEXAS syndrome and identifies two new disease causing mutations.

Key Points• Occult marrow disease is demonstrable in 68% of patients with solitary plasmacytoma of bone and is predictive of progression.• Trials of adjuvant systemic therapy are warranted in high-risk patients.The purpose of this study was to use multiparameter flow cytometry to detect occult marrow disease (OMD) in patients with solitary plasmacytoma of bone and assess its value in predicting outcome. Aberrant phenotype plasma cells were demonstrable in 34 of 50 (68%) patients and comprised a median of 0.52% of bone marrow leukocytes. With a median follow-up of 3.7 years, 28 of 50 patients have progressed with a median time to progression (TTP) of 18 months. Progression was documented in 72% of patients with OMD vs 12.5% without (median TTP, 26 months vs not reached; P 5 .003). Monoclonal urinary light chains (ULC) were similarly predictive of outcome because progression was documented in 91% vs 44% without (median TTP, 16 vs 82 months; P < .001). By using both parameters, it was possible to define patients with an excellent outcome (lacking both OMD and ULC, 7.7% progression) and high-risk patients (OMD and/or ULC, 75% progression; P 5 .001). Trials of systemic therapy are warranted in high-risk patients. (Blood. 2014;124(8):1296-1299 IntroductionSolitary plasmacytoma of bone (SPB) is characterized by localized areas of bone destruction by monoclonal plasma cells in the absence of clinical, laboratory, and radiologic features of multiple myeloma (MM). 1 Local irradiation remains the treatment of choice. United Kingdom national guidelines recommend doses of 40 to 50 Gy depending on tumor size, with a 2-cm margin, because this provides excellent local control.2 A significant proportion of patients will however progress with new lesions outside the irradiation field or, more typically, with generalized myeloma. The overall incidence of progression is 37% to 72%, with median reported times to progression (TTP) of 2 years or less. [3][4][5][6][7][8][9][10] We hypothesized that progression in SPB might occur as a result of occult disease outside of the irradiation field. To further evaluate this, we used multiparameter flow cytometry (MFC) to assess staging bone marrow (BM) samples for the presence of occult disease and determine its impact on outcome. Patients and methodsFifty patients (median age, 65years) with a histologically confirmed diagnosis of SPB according to International Myeloma Working Group (IMWG) criteria 1 were evaluated between 1998 and 2008. The solitary nature of the presenting lesion was confirmed by plain radiography in all patients, and 35 also had negative spinal magnetic resonance imaging results. Positron emission tomography (PET) scanning was not routinely performed. Thirty-five of 50 patients had a monoclonal protein (27 IgG, 7 IgA, and 1 light-chain only) with a median concentration of 7 g/L. Urine immunofixation demonstrated monoclonal urinary light chains (ULC) in 11 of 45 patients, but the serum-free light-chain (SFLC) assay was not routinely available. Patients were treated in two r...

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