Single-Tube 10-Fluorochrome Analysis for Efficient Flow Cytometric Evaluation of Minimal Residual Disease in Plasma Cell Myeloma

Abstract Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.

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Aggressive and extramedullary plasma cell myeloma evade bone marrow flow cytometric minimal residual disease detection

British Journal of Haematology ◽

10.1111/bjh.13655 ◽

2015 ◽

Vol 173 (6) ◽

pp. 947-949 ◽

Cited By ~ 1

Author(s):

Neil Came ◽

Vuong Nguyen ◽

David Westerman ◽

Simon Harrison

Keyword(s):

Bone Marrow ◽

Minimal Residual Disease ◽

Plasma Cell ◽

Residual Disease ◽

Disease Detection ◽

Plasma Cell Myeloma ◽

Minimal Residual Disease Detection ◽

Flow Cytometric ◽

Minimal Residual

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Marked Variability in Reported Minimal Residual Disease Lower Level of Detection of 4 Hematolymphoid Neoplasms: A Survey of Participants in the College of American Pathologists Flow Cytometry Proficiency Testing Program

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2014-0543-cp ◽

2015 ◽

Vol 139 (10) ◽

pp. 1276-1280 ◽

Cited By ~ 17

Author(s):

Michael Keeney ◽

Jaimie G. Halley ◽

Daniel D. Rhoads ◽

M. Qasim Ansari ◽

Steven J. Kussick ◽

...

Keyword(s):

Flow Cytometry ◽

Chronic Lymphocytic Leukemia ◽

Minimal Residual Disease ◽

Plasma Cell ◽

Myeloid Leukemia ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Lymphocytic Leukemia ◽

Plasma Cell Myeloma ◽

Minimal Residual

Context Flow cytometry is often applied to minimal residual disease (MRD) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. Objectives To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. Design Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry—Immunophenotypic Characterization of Leukemia/Lymphoma) in the spring of 2014. Results A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. Conclusions There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MRD assay's LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report.

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Flow cytometry in the diagnosis of plasma cell tumors and assessment of minimal residual disease

Oncohematology ◽

10.17650/1818-8346-2021-16-3-16-25 ◽

2021 ◽

Vol 16 (3) ◽

pp. 16-25

Author(s):

L. Yu. Grivtsova ◽

T. Yu. Mushkarina ◽

V. V. Lunin ◽

P. A. Zeynalova

Keyword(s):

Minimal Residual Disease ◽

Plasma Cell ◽

Residual Disease ◽

World Health ◽

Hematopoietic Tissue ◽

Flow Cytometric ◽

Plasma Cell Neoplasms ◽

Health Organization ◽

Minimal Residual

The article considers the features and possibilities of flow cytometric diagnostics of plasma cell neoplasms, taking into account the classification of lymphoid and hematopoietic tissue tumors of the World Health Organization, revision of 2017 and the NCCN clinical recommendations, 2021. Standardized flow cytometric protocols (the Euro-Flow conception) and algorithms for both the diagnosis of plasma cell tumors and the detection of minimal residual disease in plasma cell myeloma are described.

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Combined ROR1 and CD160 Detection For Improved Minimal Residual Disease In Patients With Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v122.21.2572.2572 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2572-2572 ◽

Cited By ~ 2

Author(s):

Timothy W. Farren ◽

Fengting Liu ◽

Marion G. Macey ◽

Thomas J. Kipps ◽

Noel Warner ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Minimal Residual Disease ◽

Research Funding ◽

Residual Disease ◽

Lymphocytic Leukemia ◽

Flow Cytometric Assay ◽

Single Tube ◽

Flow Cytometric ◽

Sequential Gating ◽

Minimal Residual

Abstract Introduction Over the past decade, treatments for patients with chronic lymphocytic leukemia (CLL) have produced complete remissions (CR) without evidence for minimal residual disease (MRD), particularly for younger and/or fitter patients. In this setting, achieving an MRD-negative CR has prognostic implications, yielding longer progression free survival (PFS) and overall survival (OS) than for patients who achieve a CR with persistent MRD. Complicating efforts to incorporate testing for MRD in clinical practice has been the lack of a defined consensus on the methods of MRD detection. In this study, we report on a novel combination of mAbs for MRD detection by flow cytometry based on two antigens: the NK-cell receptor and tumor specific antigen, CD160; and the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR-1). Objective To compare a novel single-tube, tumor-specific (CD160+ROR1) targeted approach to MRD detection against the previously published CD160 flow cytometric assay (CD160FCA) (Farren et al, 2011) and the new, single-tube 8-color ERIC assay. Methods Between October 2012 and July 2013, prospective assessment of MRD was performed on peripheral blood in 56 patients (86 samples). We developed a flow cytometric assay using mAbs specific for CD160 or ROR1 (Fukuda et al, 2008). For this we used the following mAb from BD Biosciences: CD2 FITC, CD5 Pe-Cy7, CD19 PerCP5.5, CD45V500, CD160PE, ROR-1 AF647 (“ROR-160FCA”) and a sequential gating strategy. This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished). Light chain analysis (LCR) was performed in all cases and reported where detectable. A proof of concept spiking experiment simulating MRD was prepared by mixing CLL and normal peripheral blood leukocytes in a serial dilution to a level of 10-5(n=3). Statistical analysis was performed using Spearman Rank correlation coefficients, Mann-Whitney t-test, and Bland-Altman method comparison. Significance was set at <0.05%. Results To establish the proof of principle, MRD levels ranging from 0.001% to 100% (Neat CLL) were prepared by serial dilutions, in which MRD levels could be established by the ROR-160FCA to 10-5 (n=3). Assessment of the observed incidence against expected incidence of CLL MRD demonstrated a highly significant correlation (R2=0.96, p=0.01). In the study, the range of detectable disease went from <0.01% to 38.59%, of which 37% of samples had levels below <0.01%. Analyzing all flow cytometric methods, a highly significant correlation was observed between all three: CD160FCA vs ERIC: Spearman R=0.96 (95%CI: 0.93 - 0.97, p<0.001); CD160FCA vs ROR-160FCA: Spearman R=0.97 (95%CI: 0.96 – 0.99, p<0.001); ROR-160FCA vsERIC: Spearman R=0.97 (95%CI: 0.94 – 0.98, p<0.001). 54 samples had levels of disease <1%. Bland-Altman assay comparison in these patients again demonstrated significant associations between the assays (CD160FCA vs ERIC: mean 0.08 ±0.15; CD160FCA vs ROR-160FCA: mean 0.04 ±0.19; ROR-160FCA vs ERIC: mean 0.03 ±0.25). Light chain restriction was detectable in 24 patients (size of the restricted population ranged from 0.2% to 47% of all cellular events). This sub-group of patients also demonstrated excellent correlation between level of LCR and detectable disease by CD160FCA (Spearman R=0.96, 95%CI: 0.92-0.98, p<0.001), ERIC (R=0.95, 95%CI: 0.92-0.98, p<0.01) and ROR-160FCA (R=0.97, 95%CI 0.93-0.98, p<0.001). Conclusion Monitoring minimal residual disease in CLL is a key focus for clinical trials, as MRD is an important prognostic marker in CLL in terms of PFS and OS. Here we provide a single tube assay, ROR-160FCA, which is unique by targeting two antigens restricted to malignant B-cells, CD160 and ROR1. ROR-160FCA is equivalent in MRD detection compared to both CD160FCA and the current ERIC assay under development. The two tumor-specific antigens give ROR-160FCA the potential for improved sensitivity, particularly where limited sample is available. Furthermore, it only requires a simple sequential gating strategy, is rapid, and appears more cost effective than other Methods. References Farren TW, Giustiniani J, Liu FT et al. Blood. 2011;118 (8):2174-2183. Fukuda T, Chen L, Endo T et al. Proc Natl Acad Sci U S A. 2008;105 (8):3047-3052. Disclosures: Farren: BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.

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Flow Cytometric Detection of Minimal Residual Disease In Acute Myeloid Leukemia – the Addition of hMICL and CD123 Add to Sensitivity and Applicability

Blood ◽

10.1182/blood.v116.21.2719.2719 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2719-2719

Author(s):

Anne Stidsholt Roug ◽

Jan A. Nolta ◽

Hanne Oestergaard Larsen ◽

Claus Sondergaard ◽

Peter Hokland

Keyword(s):

Acute Myeloid Leukemia ◽

Minimal Residual Disease ◽

Myeloid Leukemia ◽

Residual Disease ◽

Stable Expression ◽

Additional Tool ◽

Flow Cytometric ◽

Flow Cytometric Evaluation ◽

Minimal Residual ◽

Acute Myeloid

Abstract Abstract 2719 The outcome of acute myeloid leukemia (AML) is strongly correlated to the extent of minimal residual disease (MRD) at remission. Fusion transcripts, over-expressed genes and mutated genes have been employed in MRD quantification by RQ-PCR and are applicable for 30%–80% of all patients. More recently, flow cytometry (FCM) has been implemented as an additional tool for MRD detection employing strict gating criteria in detection of so-called leukemia associated aberrant immunophenotypes (LAIPs). In line with data from the Schuurhuis group we have identified human Myeloid Inhibitory C-type lectin (hMICL) as a candidate for AML marking at diagnosis and stable expression at relapse in a consecutive study of more than 150 patients. Importantly, we found expression of hMICL in the progenitor populations (defined as CD34+CD38- or CD117+CD38-) in AML cases. This contrasts to its absence on nearly all hematopoietic progenitors in healthy individuals and its lack of expression in acute lymphoblastic leukemia patients. Given that CD123 is also expressed in the vast majority of AML blasts at diagnosis and is characterized as a myeloid progenitor marker we hypothesized that the combination of hMICL and CD123 might serve as widely applicable MRD markers in AML and as such improve sensitivity for this approach. We performed a prospective study encompassing 38 patients with 74 follow up samples. Importantly, the assay was performed in strict parallel with other LAIPs, i.e. with no special provisions for the new markers. This FCM assay correlated strongly (88%) to available data from the most sensitive RQ-PCR marker of a series of molecular aberrations (p=0.37, McNemars chi^2 test), and that, the MRD detection and consequently reduced qualitative discordance resulted from a fourfold reduction of patients with FCM negative results but positive RQ-PCR result (table 1). The quantitative correlation coefficient between RQ-PCR and FCM increased from 0.46 to 0.7 by addition of hMICL and CD123 (figure 1). When sensitivities of the FCM assay were directly compared to those of validated RQ-PCR assays it was apparent that that of the FCM was inferior. However, it was still in the range in which it strongly predicted treatment failure. Undoubtedly, running more cells and samples and by employing more stringent gating and post-run analyses will further improve this strategy. However, given that treatment failures in AML are apparent by WT1 RQ-PCR with sensitivities at the 1; 1.000 range, the standard hMICL/CD123 assay should suffice in the vast majority of situations. We conclude that high-density expression of both hMICL and CD123 in the vast majority of AML cases at diagnosis and stable expression during the course of the disease combined with high sensitivity holds the potential for wide applicability and improved MRD monitoring by FCM using setup and interpretation not requiring special attention. This assay promises to be of great value in fast and efficient management of all AML patients. Table 1. Qualitative concordance between RQ-PCR, rFCM* and tFCM# MRD results RQ-PCR positive patients Samples RQ-PCR+ FCM+ RQ-PCR- FCM- RQ-PCR+ FCM- RQ-PCR- FCM+ Concordance McNemars chi2 rFCM Vs RQ-PCR 43 16 8 16 3 24/43(56%) p=0,044 tFCM Vs RQ-PCR 43 28 10 4 1 38/43 (88%) p=0,37 RQ-PCR negative patients Samples rFCM+ tFCM+ rFCM− tFCM− rFCM+ tFCM− rFCM−/tFCM+ Concordance ND¤ rFCM Vs tFCM 23 13 0 8 2 13/23(56%) ND¤ * *Routine panel of antigens in routine flow cytometric evaluation of MRD # Test panel of antigens in flow cytometric evaluation of MRD including CD123, CD34 and hMICL ¤ Not done. Figure 1. Correlations between RQ-PCR and FCM Figure 1. Correlations between RQ-PCR and FCM Disclosure: No relevant conflicts of interest to declare.

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