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Introduction: The diagnosis of CLL/SLL relies on flow cytometric immunophenotyping. Increasing emphasis is being placed on precise detection of the minimal residual disease. Following antigen recommendations of ERIC and ESCCA's Harmonization Project, we validated a 14-color assay for the characterization CD5+ lymphoproliferative neoplasms and CLL MRD with a sensitivity of at least 10 -4 ., Methods: The assay was designed based on ERIC/ESCCA recommended antigens with the addition of CD40 for alternate gating when CD19 expression is reduced. Lower limit of quantitation/lower limit of detection, assay procedural precision, linearity, and limit of blank were established. Then, 52 CD5+ B-cell lymphoproliferative neoplasms (41 CLL/11 non-CLL) and 29 normal samples were used for parallel evaluation. Automated cluster identification and quantitation of CLL clones in MRD setting was performed using Barned-Hutt SNE. Separation analysis between CLL and non-CLL phenotypes was performed by PCA and bh-SNE., Results: Separation ratios for each antigen exceeded ERIC/ESCCA guidelines. Precision was <20% at LLOQ (0.01%). The limit of blank was <10/500,000 cells. Concordance between the 14-color and legacy assay (Deming regression y = 1.01x, r 2  = .99) was seen. All 20 samples with MRD levels 0.5%-0.006% (median 0.04%) showed an abnormal cell cluster by bh-SNE, with concordant results between manual and automated quantitation (y = x, r 2  = 1). CLL cases clustered together and away from mantle cell lymphoma by bh-SNE and PCA with outlier atypical phenotype CLL cases posing diagnostic challenges by both manual and automated analysis., Conclusion: The 14-color CD5+ LPD assay provides a robust standardization platform for MRD and disease characterization using both manual and automated analysis., (© 2020 International Clinical Cytometry Society.)

The 2016 International Myeloma Working Group consensus recommendations emphasize high-sensitivity methods for minimal residual disease (MRD) detection, treatment response assessment, and prognostication. Next-generation sequencing (NGS) of IGH gene rearrangements is highly specific and sensitive, but its description in routine clinical practice and performance comparison with high-sensitivity flow cytometry (hsFC) remain limited. In this large, single-institution study including 438 samples from 251 patients, the use of NGS targeting the IGH and IGK genes for clonal characterization and monitoring, with comparison to hsFC, is described. The index clone characterization success rate was 93.6% (235/251), which depended on plasma cell (PC) cellularity, reaching 98% when PC ≥10% and below 80% when PC <5%. A total of 85% of cases were successfully characterized using leader and FR1 primer sets, and most clones showed high somatic hypermutation rates (median, 8.1%). Among monitoring samples from 124 patients, 78.6% (147/187) had detectable disease by NGS. Concordance with hsFC was 92.9% (170/183). Discordant cases encompassed 8 of 124 hsFC MRD+/NGS MRD- patients (6.5%) and 4 of 124 hsFC MRD-/NGS MRD+ patients (3.2%), all with low-level disease near detection limits for both assays. Among concordant hsFC MRD-/NGS MRD- cases, only 5 of 24 patients (20.8%) showed subsequent overt relapse at 3-year follow-up. HsFC and NGS showed similar operational sensitivity, and the choice of test may depend on practical, rather than test performance, considerations., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) may soon replace routine electrophoretic methods for monitoring monoclonal proteins in patients with multiple myeloma. To further evaluate the clinical utility of this assay, we compared the performance of MALDI-TOF-MS head-to-head with an established bone marrow-based measurable residual disease assay by flow cytometry (Flow-BM-MRD), using Memorial Sloan Kettering Cancer Center's 10-color, single-tube method. Our results suggest that MALDI-TOF-MS adds value to bone marrow-based MRD testing and may be most useful for early detection of relapse in peripheral blood compared to current electrophoretic methods., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Recent years saw significant breakthroughs in treatment of multiple myeloma. Durable remissions are now seen in a significant proportion of patients with the previously uniformly incurable and progressive disease. Yet because of deep suppression of the neoplastic myeloma clones by the newer therapies, older disease monitoring techniques are insufficient to distinguish between the patients at high risk of imminent relapse and those in whom durable remission is expected. This review briefly describes prognostic and therapeutic implications of measurable disease (MRD) evaluation, explains why deep MRD evaluation is needed for patients without morphologic evidence of disease, and reviews the state of the art of evaluation of myeloma MRD by flow cytometry., Competing Interests: Declaration of competing interest The author has received consulting fees from Celgene for a project that was not in connection with myeloma therapy or monitoring., (Copyright © 2020. Published by Elsevier Ltd.)

The outlook for myeloma patients has steadily improved with the introduction of newer drug combinations in recent years. Unlike older therapies that largely achieved only modest levels of neoplastic clone reduction, the newer drug combinations have led to deeper suppression of myeloma clones in most patients. Frequently the neoplastic clones become undetectable with traditional disease evaluation approaches. Recent studies using ultrasensitive disease monitoring have demonstrated that patients with disease undetectable by traditional techniques show wide heterogeneity in disease levels varying by several orders of magnitude. Moreover, measurement of the depth of disease suppression even at very low level has emerged as the most powerful prognostication tool in myeloma. Minimal (or measurable) residual disease (MRD) evaluation has also been proposed as a relevant tool in assessment of drug efficacy and in selection of further therapeutic options. In the face of the robust MRD measurement utility data, it has become critical to develop widely applicable disease monitoring techniques that can be applied to more patients in a variety of clinical setting. Both DNA-based and flow cytometry-based approaches have been successfully developed for this purpose achieving sensitivity approaching 1 neoplastic cell in a million. This review article focuses on the theoretical and practical aspects and challenges of deep MRD monitoring in myeloma by flow cytometry. Challenges of flow cytometric disease monitoring in the era of antigen-directed therapy are also discussed., (Copyright © 2018. Published by Elsevier Inc.)

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Objectives: Widespread adoption of recent recommendations for minimal residual disease (MRD) detection in myeloma has partly been impeded by a paucity of studies detailing multiparameter flow cytometry (MPF) assay validation. In response, we have validated a novel and efficient single-tube 10-color assay for MRD detection that incorporates the recently recommended plasma cell markers., Methods: Aspirate samples from 53 patients with plasma cell disorder were analyzed using a novel single-tube 10-color method. The limit of detection, precision of measurement, and linearity of measurement of our new assay were determined using serial dilution experiments. The stability of the new antibody cocktail and the viability/specificity of stained samples were evaluated using serial time course measurements., Results: There was a high degree of quantitative agreement between our new 10-color method and an established eight-color method. Four positive samples detected by the 10-color assay were below or at the limit of detection of the eight-color assay, confirming its high sensitivity. In two cases, subsequent revision of the International Myeloma Working Group Uniform Response Criteria was necessary., Conclusion: Adoption of our validated 10-color assay would enable clinical laboratories to satisfy current MRD recommendations without significantly increasing the demands on current workflow practices., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)