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

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).

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.

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 This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished).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%.

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).

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.

Farren:BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.