Identification of Atypical Plasma Cells in Patients with MGUS or Multiple Myeloma Through Expression of Immunoreceptor CD229

While multiparameter flow cytometry (MPF) is an integral part in the diagnosis, disease staging and response evaluation for hematologic malignancies such as acute leukemia or non-hodgkin-lymphoma, MPF for multiple myeloma (MM) is still mostly restricted to research studies or only performed by specialised laboratories experienced in the technique of immunophenotyping. Furthermore, the exact phenotype of malignant myeloma cells is still a matter of debate. Recently, we have identified CD229, a surface marker belonging to the family of signaling lymphocytic activation molecules (SLAM) involved in lymphocyte activation as a potential novel target for diagnosis and treatment of MM. CD229 is expressed on freshly isolated myeloma cells including their clonogenic precursors and several myeloma cell lines. In order to further validate our findings from a previous pilot study, we now analysed 151 samples from 81 patients with suspected or proven MM or monoclonal gammopathy of uncertain significance (MGUS) via MPF.

Between May 2010 and May 2012, specimens (bone marrow (n=142), peripheral blood (n=10), cells from isolated plasmocytoma (n=1)) from patients (pts) with MM (n=65), plasmocytoma (n=1), MGUS (n=6), lymphoplasmacytic lymphoma (n=1) and patients with suspected MM (n=8) were simultaneously analysed via cytology and 8-colour MPF. 19 pts. were analysed at least 3 times during the course of their disease so that CD229 expression could be followed under therapy. Plasma cells were specified using surface markers CD38, CD138, CD45 and cytoplasmatic light chain restriction. Antigens analysed on plasma cells were CD19, CD28, CD33, CD56, CD81, CD117, CD200, CD221 and CD229.

Although plasma cell numbers determined by MPF were constantly considerably lower compared to simultaneously determined cytology results, linear regression model showed a highly significant correlation between plasma cell percentages in bone marrow measured by MPF with cytology (p<0. 0001). Plasma cell enumeration in pB also showed a strong correlative trend between cytologic and MPF results, however, due to lower numbers (n=10), this was not statistically significant (p = 0. 057).

CD229 could be detected on all atypical plasma cells irrespective if they were found in MGUS or MM samples. The median of mean fluorescence intensity (MFI) of CD229 expression on plasma cells was 3, 63 (range −144. 1 – 34, 23). Median MFI on MM samples (3, 62; range −144 − 34, 23; n=131) did not differ from MFI on atypical plasma cells in pts with MGUS (3, 74, range 1. 07 – 8, 65; n=9). CD229 expression was highest on atypical plasma cells with expression of CD56 compared to CD56 negative plasma cells (p<0. 0001). This was confirmed when correlation of marker expression was evaluated. CD229 expression was clearly correlated with expression of CD56 (n=141, p = 0. 03), CD117 (n=139, p = 7E–08) and CD200 (n=140; p = 0. 03), while it was inversely correlated with expression of CD19 (n=140; p = 0. 03). Serial CD229 expression (>= twice) was determined in 39 patients. Except for three samples, where plasma cell counts became less than 1% of bone marrow cells, CD229 expression remained stable throughout the various analyses.

While the exact function of the immunoreceptor CD229 on myeloma cells is still unknown, CD229 allows identification of atypical plasma cells by MPF. Our results show that CD229 is constantly expressed on atypical plasma cells independent of therapy and can be used in addition to other surface markers for determination of malignant plasma cell phenotype and to monitor minimal residual disease (MRD) under treatment.

No relevant conflicts of interest to declare.