Validation of a modified pre‐lysis sample preparation technique for flow cytometric minimal residual disease assessment in multiple myeloma, chronic lymphocytic leukemia, and B‐non Hodgkin lymphoma

Abstract ROR1 is an oncofetal protein expressed in chronic lymphocytic leukemia (CLL). We generated a monoclonal antibody (mAb) specific for ROR1 (4A5) and used this to stain blood or marrow cells from patients with CLL and other adults. We found that 4A5 reacted with the CLL cells of all patients examined and also with some cells that had an immunophenotype of B lymphocyte precursors (BLP) (CD10+, CD19+, CD20-variable, dim CD45+, surface immunoglobulin-negative). 4A5 showed no reactivity with other blood or marrow cells. We speculated that this mAb could be used to detect CLL cells in blood or marrow of patients after therapy, providing a means with which to assess minimal residual disease (MRD). For this purpose, we evaluated a four-color combination of mAbs using CD10-FITC, CD19-PE, CD5-PerCP-Cy5.5, and 4A5-Alexa-647. MRD detection limits were established through reconstitution studies in which we made serial dilutions of CLL cells into normal blood or marrow samples. MRD was measured by whole blood lysis and acquisition of 100,000 events using a FACSCalibur and a gating strategy designed to exclude CD5-negative, CD10-positive, and 4A5-negative B lymphocytes (CD19+). CLL cells stained with 4A5 had a mean fluorescence intensity (MFI) of 31 (N=100) and a MFI ratio (MFIR) relative to that of isotype control stained CLL cells of more than 10:1. On average, 94% of the CLL cells in each sample had higher fluorescence intensity when stained with 4A5-Alexa-647 than when stained with an isotype-control mAb (range 88–98%). Marrow samples (N=9) from adults with diagnoses other than CLL and that had 1–10% BLP had CD5− negative, CD10+, CD19+ cells that reacted with the 4A5-Alexa 647. These cells in such samples had a MFI of 19 and a MFIR relative to that of isotype-control-stained BLP of 6.3. On average, 48% of such cells in each sample had higher fluorescence intensity when stained with 4A5-Alexa-647 than when stained with an isotype-control mAb (range 18–79%). Four color flow cytometric analysis with CD10, CD19, CD5, and 4A5 detected CLL cells present at less than 0.1% in reconstituted blood or marrow samples, including marrow with 3–5% BLP. Background was between 0.01% and 0.1% with whole blood (marrow) lysis when we acquired a total of 100,000 flow-cytometric events. These data indicate that anti-ROR1 mAbs can be used with mAbs specific for CD10, CD19, and CD5 for sensitive detection of MRD in CLL.

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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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Inter-Laboratory Assessment of Minimal Residual Disease In Chronic Lymphocytic Leukemia – Evaluation of the Novel CD160FCA Assay

Blood ◽

10.1182/blood.v116.21.2723.2723 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2723-2723

Author(s):

Timothy Farren ◽

Fengting Liu ◽

Claire Stephens ◽

Marion Macey ◽

Michael Jenner ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Lymphocytes ◽

Light Chain ◽

Residual Disease ◽

Lymphocytic Leukemia ◽

Cell Numbers ◽

Flow Cytometric ◽

Highly Sensitive ◽

B1 Cells ◽

Minimal Residual

Abstract Abstract 2723 With the advent of highly effective chemo-immunotherapy regimens, the treatment of chronic lymphocytic leukemia (CLL) is no longer simple palliation but aims to achieve a minimal residual disease (MRD)-negative remission. Historically, CD5 and CD19 with demonstration of clonality was the mainstay of MRD evaluation in CLL. However, this approach is limited by the presence of normal B1-cells (CD5+CD19+) and the inability to demonstrate light-chain restriction with very low B-cell numbers. More recently, an international standardized approach (ISA) to the assessment of MRD in CLL was published with a detection accuracy of 95.7% for the detection CLL above 0.01%, involving 8 monoclonal antibodies (mAbs) in a 5 test system.1 CD160 is an activatory NK cell receptor, but is not expressed on normal B-lymphocytes, B1-cells, naive B-cells or activated B-lymphocytes. CD160 is expressed on malignant B-cells in >98% of cases of CLL, but in only 15% of non-CLL cases. This makes CD160 a unique marker for the assessment of MRD. Methods: Utilizing the anti-CD160 monoclonal antibody (BY55, Coulter Immunotech, Marseille, France), we have developed a highly sensitive CD160 Flow Cytometric Assay (CD160FCA) incorporating CD2, CD5, CD19, CD23 and CD160. Whole blood (1×106 leukocytes) were labeled with mAbs, followed by an ammonium chloride-based lysing method. Data acquisition was captured using a sequential gating strategy focusing only on the malignant population. The CD160FCA was evaluated in two centers and the results compared with the ISA methodology in two different centers. Results: As expected, light-chain restriction could not be reliably measured in patients with very low B-cell numbers. 23 cases were analyzed at four centers. Concordant results were found in 22/23 cases with a sensitivity of 1.00 (P=0.0023, OR 91.00). Of the 23 patients 87% were MRD positive by the CD160FCA compared to 82% by the ISA. There were no cases that were MRD positive by the ISA and negative by the CD160FCA. There was a close correlation between the two methodologies for disease levels above 0.01% (Spearman Rank R=0.9913, P<0.0001). When studying the median fluorescence intensity (MFI) of CD160, the MRD positive cases had a mean MFI of 374 (288-461 95%CI) and the negative cases 47 (2-93 95%CI) (Figure 2). The CD160 FCA offers a single tube analysis for MRD, which is highly sensitive, independent of the therapy and can be employed throughout treatment. Importantly, not only is this approach simpler, cheaper and faster than current MRD approaches, it is still informative in cases where no light chain clonal population is found, or where restriction studies have failed. With increasing evidence that MRD detection in CLL can predict patient outcome, CD160FCA represents a simple tool for MRD assessment. Reference: Rawstron AC, Villamor N, Ritgen M et al. International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia. Leukemia. 2007;21(5):956-964. Disclosure: No relevant conflicts of interest to declare.

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