Using CD160 as a Novel Approach to the Assessment of Minimal Residual Disease (MRD) in Chronic Lymphocytic Leukaemia (CLL).
Authors: Farren Timothy , Giustiniani Jerome , Johansson Ulrika , Lim Christina , Athorn Gemma , Newland Adrian , Macey Marion , Agrawal Samir 
Published: April 2020

Abstract
Despite recent therapeutic advances, B-cell CLL (CLL) remains an incurable disease. Achieving MRD negativity in CLL has been associated with improved outcomes. Historically, co-expression of CD5 and CD19 with kappa/lambda light-chain (LC) restriction has been the mainstay of evaluating MRD in CLL. Using both 2 and 4-colour flow cytometry, interpretation was still complicated by the presence of normal CD5+ B cells and the difficultly of establishing LC restriction, as well as its significance, at very low levels of B-cells. CD160 is an activatory NK cell receptor also found on a subset of cytotoxic T-cells. CD160 is not expressed on normal B-lymphocytes or myeloid cells. We have previously reported that CD160 is aberrantly expressed on malignant B-cells, including &gt;98% of CLL cases1,2. Using anti-CD160 monoclonal antibody (BY55, Coulter Immunotech, Marseille, France), we have developed a highly sensitive multi-colour, single tube flow cytometric (MC-ST-FC) assay. Whole blood (1×106 leukocytes) were labelled with antibodies, followed by a lysed/washed approach using an ammonium chloride-based lysing and fixing methodology, and analysed on a FACS Canto (Becton Dickinson). In excess of 10,000 events were recorded within the ‘disease gate’ and MRD assessed by CD19+/CD160+/CD5+/CD2-/CD3- co-expression was compared with CD5+/CD19+ co-expression and LC restricted analysis. Dilution experiments showed the CD160 MC-ST-FC assay had a sensitivity of at least 104. PCR for CD160 mRNA was performed on highly purified B-cells (&gt;98% CD19+ using magnetic beads). 98 samples from 36 patients were assessed by two and multi-colour flow cytometry at specified time points throughout their treatment, using a cut-off limit of 1% for CD19/CD160 and 10% for CD5/CD19 co-expression. 30 blood samples were analysed for CD160 expression using PCR. LC restriction could not be reliably measured in patients with very low levels of B-cells. Of 55 samples with &lt;10% CD5/CD19 co-expression, 41 samples did not show LC restriction. 31 of these cases had low level CD19/CD160 expression (2 to 10%) indicating MRD positivity - these cases would otherwise have erroneously been called MRD negative. In 21 cases, &lt;10% CD5/CD19 expression with LC analysis (6 with normal κ/λ, 8 with insufficient cells) and CD19/CD160 (with 7 cases of equivocal 1% CD19/CD160 co-expression) gave MRD negative results. These were subsequently confirmed with PCR. The remaining 37 samples showed evidence of residual disease using both &gt;10% CD5/CD19 and &gt;2% CD19/CD160 expression, again confirmed by PCR. Qualitative PCR results were in direct concordance with the flow assay. There were no samples in which CLL cells were detected by PCR, but not by the CD160 MC-ST-FC assay. CD160 MC-ST-FC assay is a sensitive assay for CLL MRD, with better performance than dual CD5/CD19 with LC analysis. CD160 expression is stable during, and post-therapy and is applicable to vitually all cases of CLL. There was excellent correlation with PCR. Furthermore, the CD160 MC-ST-FC assay is simpler, faster, cheaper and applicable to more patients, than current molecular and flow cytometric methods.
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