This International Myeloma Working Group consensus updates the disease defi nition of multiple myeloma to include validated biomarkers in addition to existing requirements of attributable CRAB features (hypercalcaemia, renal failure, anaemia, and bone lesions). These changes are based on the identifi cation of biomarkers associated with near inevitable development of CRAB features in patients who would otherwise be regarded as having smouldering multiple myeloma. A delay in application of the label of multiple myeloma and postponement of therapy could be detrimental to these patients. In addition to this change, we clarify and update the underlying laboratory and radiographic variables that fulfi l the criteria for the presence of myeloma-defi ning CRAB features, and the histological and monoclonal protein requirements for the disease diagnosis. Finally, we provide specifi c metrics that new biomarkers should meet for inclusion in the disease defi nition. The International Myeloma Working Group recommends the implementation of these criteria in routine practice and in future clinical trials, and recommends that future studies analyse any diff erences in outcome that might occur as a result of the new disease defi nition.

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International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma.

Treatment of multiple myeloma has substantially changed over the past decade with the introduction of several classes of new effective drugs that have greatly improved the rates and depth of response. Response criteria in multiple myeloma were developed to use serum and urine assessment of monoclonal proteins and bone marrow assessment (which is relatively insensitive). Given the high rates of complete response seen in patients with multiple myeloma with new treatment approaches, new response categories need to be defined that can identify responses that are deeper than those conventionally defined as complete response. Recent attempts have focused on the identification of residual tumour cells in the bone marrow using flow cytometry or gene sequencing. Furthermore, sensitive imaging techniques can be used to detect the presence of residual disease outside of the bone marrow. Combining these new methods, the International Myeloma Working Group has defined new response categories of minimal residual disease negativity, with or without imaging-based absence of extramedullary disease, to allow uniform reporting within and outside clinical trials. In this Review, we clarify several aspects of disease response assessment, along with endpoints for clinical trials, and highlight future directions for disease response assessments.

https://www.thelancet.com/pdfs/journals/lanonc/PIIS1470-2045(16)30206-6.pdf

International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.

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EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.

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EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

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.

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Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

1 Sirohi B, Powles R. Multiple myeloma. Lancet 2004; 363: 875–887. 2 Zipori D, Duksin D, Tamir M, Argaman A, Toledo J, Malik Z. Cultured mouse marrow stromal cell lines. II. Distinct subtypes differing in morphology, collagen types, myelopoietic factors, and leukemic cell growth modulating activities. J Cell Physiol 1985; 122: 81–90. 3 Belaid Z, Hubint F, Humblet C, Boniver J, Nusgens B, Defresne MP. Differential expression of vascular endothelial growth factor and its receptors in hematopoietic and fatty bone marrow: evidence that neuropilin-1 is produced by fat cells. Haematologica 2005; 90: 400–401. 4 Vanderkerken K, Asosingh K, Willems A, De Raeve H, Couck P, Gorus F et al. The 5T2MM murine model of multiple myeloma: maintenance and analysis. Methods Mol Med 2005; 113: 191–205. 5 Kumar S, Greipp PR, Haug J, Kline M, Chng WJ, Blood E et al. Gene expression profiling of myeloma cells at diagnosis can predict response to therapy with thalidomide and dexamethasone combination. ASH Annual Meeting Abstracts 2005; 106: 508. 6 Vanderkerken K, De Greef C, Asosingh K, Arteta B, De Veerman M, Vande Broek I et al. Selective initial in vivo homing pattern of 5T2 multiple myeloma cells in the C57BL/KalwRij mouse. Br J Cancer 2000; 82: 953–959. 7 Alexandrakis MG, Passam FH, Sfiridaki A, Pappa CA, Moschandrea JA, Kandidakis E et al. Serum levels of leptin in multiple myeloma patients and its relation to angiogenic and inflammatory cytokines. Int J Biol Markers 2004; 19: 52–57. 8 Gimble JM, Robinson CE, Wu X, Kelly KA. The function of adipocytes in the bone marrow stroma: an update. Bone 1996; 19: 421–428.

Carine Seynaeve

Papers

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

Janus kinase mutations in the development of acute megakaryoblastic leukemia in children with and without Down’s syndrome