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Background: Multicolor flow cytometry (MFC) is increasingly important for the diagnosis and minimal residual disease (MRD) assessment of patients with plasma cells (PC) dyscrasias, like multiple myeloma. Recently published information shows that immunophenotype of myeloma PC can change over time and normal PC are heterogeneous in the expression of CD19 and CD56. This implies that for a sensitive, reliable detection of MRD clonality assessment by the detection of cytoplasmic kappa and lambda light chains is advisable., Methods: Eight-color MFC was used to detect normal and myeloma PC by the expression of CD38 and CD138. Analysis of additional surface antigens like CD45, CD19, CD56, CD27, and the intracellular immunoglobulin light chain distribution were used to differentiate polyclonal from clonal PC., Results: Absence of cytoplasmic light chains expression in a PC subpopulation with an abnormal phenotype suggested the presence of non-secretory plasma cells in the bone marrow (BM) of this patient. This observation however, was contradicted by the presence of free lambda light chains in the patient's serum. After repeating the analysis with polyclonal antibodies against intracellular immunoglobulin light chains instead of monoclonal antibodies, the abnormal PC subpopulation appeared to express lambda light chains., Conclusion: These data illustrate that if clonality assessment of PC is included in disease monitoring, the use of polyclonal over monoclonal antibodies is preferred for the detection of intracellular immunoglobulin light chains., (Copyright © 2012 International Clinical Cytometry Society.)

Cytotoxic T cells play an important role in graft-versus-host-disease (GvHD) and graft-versus-leukaemia/myeloma, which may occur in patients treated with an allogeneic stem cell transplantation (ASCT). Here, we describe the selection of a myeloma reactive CD4+ cytotoxic T cell-line (CTL) and two CD4+ clones from this CTL. The CTL was generated from the blood from a patient with multiple myeloma (MM) with graft versus myeloma/GvHD, following an ASCT. The CTL was stimulated using irradiated peripheral blood mononuclear cells and EBV transformed B cells from the myeloma patient (EBVp), both of which were obtained prior to ASCT. Both the CTL and the two T cell clones specifically lysed EBVp and secreted IFN-gamma after coculture with EBVp and autologous myeloma tumour cells in a class II restricted fashion. These results show that myeloma tumour cells and autologous B cells present a common polymorphic peptide that functions as a target for graft derived cytotoxic T cells. Identification of these proteins will give insight into the relationship between graft versus myeloma (GvM) and GvHD and may provide immunotherapeutical targets in the treatment of MM.

An antisense oligodeoxynucleotide (ODN) complementary to the first six codons of the Bcl-2 mRNA, G3139 (oblimersen sodium; Genasense), has been shown to downregulate Bcl-2 and produce responses in a variety of malignancies including drug-resistant lymphoma. Incubation of ex vivo purified plasma cells from patients with multiple myeloma (MM) with carboxyfluorescein (FAM)-labeled antisense ODNs resulted in a time- and dose-dependent uptake in the cytoplasm and nucleus. No major differences in uptake of Bcl-2 antisense ODNs were observed among patients' samples. Incubation of purified myeloma plasma cells with G3139, but not solvent or reverse polarity control ODNs, resulted in a reduction (>75%) of Bcl-2 mRNA levels after 2 and 4 days, as measured by Real-Time PCR. Treatment with G3139 led to a sequence-specific reduction of Bcl-2 protein levels within 4 days of exposure in 10 out of 11 clinical samples from patients with chemosensitive and multidrug-resistant disease, without significant reduction of alpha-Actin, Bax, Bcl-XL, or Mcl-1 proteins. This resulted in a significantly enhanced sensitivity of the myeloma tumor cells to dexamethasone or doxorubicin-induced apoptosis. G3139 can consistently enter myeloma cells, downregulate the expression of Bcl-2, and enhance the efficacy of myeloma therapy. These data support further clinical evaluation of G3139 therapy in multiple myeloma.

Expression of CD44v9-containing isoforms (CD44v9) on myeloma plasma cells correlates with unfavorable prognosis, suggesting that CD44 variant molecules are involved in the disease process. In this study, the presence of CD44v on B cell lines from different stages of development was analyzed by flow cytometry and a role in adhesion to stromal cells from different tissues was evaluated in in vitro binding assays. CD44v3, v6 and v9 isoforms were exclusively expressed on plasma cell lines and CD44v9 expression correlated with IL-6-dependent plasma cell growth. Binding studies using CD44 isoform- specific reagents showed that CD44v6 and CD44v9 were involved in binding to bone marrow stromal cells, but not to in vitro synthesized ECM or hyaluronic acid. CD44v9-mediated plasma cell binding resulted in a significant induction of IL-6 secretion by bone marrow stromal cells. Large differences in quantitative plasma cell binding to stromal cells from different tissues were observed. These, however, could not be related to a differential use of CD44v in these binding processes. The role of CD44v9 in adhesion induced IL-6 secretion and its preferential expression on IL-6-dependent plasma cell lines may explain the previously observed correlation between CD44v9 expression and adverse prognosis in multiple myeloma.

CD44 variant isoforms (CD44v) have been shown to be important factors in adverse prognosis in hematological malignancies. To investigate whether CD44 expression is associated with malignant transformation in multiple myeloma, RNA and protein expression of CD44 standard (CD44s) and CD44v4, v6, v9, v10 containing isoforms was compared on bone marrow plasma cells from normal individuals and myeloma patients at different stages of disease. CD44s protein expression is strongly decreased on myeloma plasma cells and non-malignant B cells in affected bone marrow of myeloma patients, while no differences in CD44s expression were found between blood B cells from normal individuals and myeloma patients. CD44v isoforms were expressed on plasma cells in the majority of normal and myeloma samples analyzed. CD44v9 and v10 containing isoforms were differentially expressed on bone marrow plasma cells from normal individuals (predominantly CD44v9+v10+) and myeloma patients with stable (predominantly CD44v9-v10+) or progressive (predominantly CD44v9+v10- disease. Normal and myeloma plasma cells contained CD44 mRNA transcripts consisting of multiple CD44v exons. In addition, CD44v9 positive myeloma cells carried large CD44 transcripts. These results imply that detection of CD44v isoforms may be a valuable diagnostic tool for monitoring myeloma disease progression and response to treatment.

The monoclonal B cells present in the blood of three patients with Waldenstrøm's macroglobulinaemia were analysed according to phenotype and function. As a marker for the monoclonal nature of the detected immunoglobulin (Ig) molecules, the kappa/lambda-ratio (two patients) or the presence of idiotypic (Id) determinants (one patient) were used. With double immunofluorescence it was shown that the neoplastic blood B cells were heterogeneous in respect to maturation stage. In addition to B cells carrying membrane bound IgM and IgD (mIgM+/IgD+), both mIgM+/IgD- and cytoplasmic IgM+ blastoid cells could be detected in the blood of the patients. This heterogeneity was also reflected in the responses of the monoclonal B cells upon stimulation with mitogens and T cell factors. B blastoid cells and IgM secretion could be induced after in vitro culture in the presence of Staphylococcus aureus, Pokeweed mitogen or a combination of both mitogens. It was shown that also T cell factors were effective in inducing monoclonal B cell differentiation.