CD66a (CEACAM1) is the only CD66 variant expressed on the surface of plasma cells in multiple myeloma: a refined target for radiotherapy trials?

The membrane protein CD66 is a member of the carcinoembryonic antigen (CEA) and immunoglobulin superfamily. CD66 proteins are expressed as a number of isoforms, also termed CEACAMs (CarcinoEmbryonic Antigen Cellular Adhesion Molecule), which have a wide range of biologically important functions including cell adhesion, cellular migration, pathogen binding and activation of signalling pathways. The CD66 isoforms are derived as mRNA splice and transcriptional variants from the CEACAM gene cluster on chromosome 19q13.1-13.2. CD66 isoforms a-d have been reported to be present on myeloid lineage cells with increasing density of expression from the promyelocyte through to mature granulocytes (Hammarstrom, 1999; Nair & Zingde, 2001) with CD66a, b, c and d equivalent to CEACAM1, 8, 6 and 3, respectively. The expression of CD66 isoforms on myeloid lineage cells in the bone marrow can be exploited as targets for therapy. Phase I and II clinical trials at our centre (Orchard et al, 2006) and others (Ringhoffer et al, 2005; Zhang & Gopal, 2008) utilize this property for the delivery of targeted radiotherapy to the bone marrow as part of the conditioning regimen for transplantation in acute leukaemias and multiple myeloma. Previous studies revealed the abnormal expression of CD66 isoforms on leukaemia and solid tumour cells (Luo et al, 1999; Kang et al, 2007; Lasa et al, 2008; Ratei et al, 2008). Indeed, we have shown the presence of CD66 antigen on normal and malignant plasma cells (Richardson et al, 2005). However, little is known about the differential expression pattern of the CD66 variants on plasma cells from patients with multiple myeloma. We performed flow cytometry on two human myeloma cell lines (U266 and ARH77) and on plasma cells from patients with multiple myeloma (Table I). Fresh bone marrow samples were obtained from patients attending Southampton General Hospital between 2007 and 2008. Mononuclear cells expressing CD138, a specific plasma cell marker, were either positively selected (EasySep magnetic separation kit) or mononuclear cells were isolated and incubated with saturating amounts of CD138 antibody (Dako UK Ltd., Cambridgeshire, UK) and a single CD66 monoclonal antibody (either CD66a: R&D Systems Europe Ltd., Abingdon, UK, CD66b:GENOVAC GmbH, Freiburg, Germany, CD66c: Santa Cruz Biotechnology Inc., Heidelberg, Germany CD66d: R&D or CD66e: AbD Serotec, Oxford, UK) to examine the expression of each CD66 isoform in every patient sample. Appropriate isotype-matched controls (AbD Serotec) were used in all experiments. Data acquisition and analysis were performed using the FACScalibur cellquest Software (Becton Dickinson UK Ltd., Oxford, UK). Expression of CD66a, but not CD66b, c, d or e, was identified on both myeloma cell lines analysed (Fig 1A) and in all five clinical bone marrow specimens (purified plasma cells or CD138 gated cells)(Figs 1B, C). Positive expression of CD66a ranged from 69% to 100% of the plasma cells with a median fluorescence ranging from 54 to 673, with no detectable expression of any of the other isoforms of CD66 (Fig 1C). Positive but weaker expression was seen with utilization of the pan CD66 antibody (CD66a/b/c/e) in primary samples (Fig 1C) and the cell lines (Fig 1A). CD66 expression on multiple myeloma cell lines and patient plasma cells. The expression of all CD66 isoforms (CD66a/b/c/d/e) were examined. Expression of (A) CD66a, but not CD66b, c, d or e were found on both of the multiple myeloma cell lines analysed (U266 and ARH77). Positive but weaker expression was seen when using the pan CD66 antibody (CD66a/b/c/e) as compared to staining with the CD66a antibody; (B) CD138 and CD38 cells were gated (R1 – red population) from within the peripheral blood mononuclear cells and (C) only expression of CD66a, but no other CD66 isoform, was detected on the plasma cells analysed from five multiple myeloma patients. Again, positive but weaker expression was observed when using the pan CD66 antibody (CD66a/b/c/e), as compared to the CD66a antibody alone. We have shown for the first time the expression of CD66a but no other CD66 isoforms on multiple myeloma. These findings may help in the optimization of future radio-immunotherapeutic strategies by supporting the use of a monoclonal CD66a antibody for targeted radiotherapy in patients with multiple myeloma. C.L. is funded by a European Group for Blood and Marrow Transplant fellowship, B.G. by Leukaemia Research Fund, S.E.B. by Cancer Research U.K.