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The recent advances in myeloma treatment result in significantly better outcomes, defined as increased progression free survival (PFS) and overall survival (OS). Since there is a proven correlation between the extend of response and prolonged survival, there is an urgent need for highly sensitive assays for the detection of minimal residual disease (MRD). Next generation flow cytometry has become a valuable approach for sensitive evaluation of the depth of complete response (CR). Here, we report the diagnostic performance and validation results of a single-tube 9-color panel assay. The validation design included intra-assay analysis measuring accuracy, inter-assay analysis estimating method's linearity and precision and inter-assay analysis evaluating repeatability. Furthermore, in inter-operator analysis assessed the comparability of the result analysis of different operators. Staining stability was evaluated in age-of-stain experiments. Our validation results show that a reliable detection of residual myeloma cells is feasible to a detection level of 10 -5 with a single-tube assay for a variety of materials (peripheral blood, bone marrow and stem cell apheresis). This study establishes highly sensitive, fully standardized approach for MRD detection in myeloma that is ready for implementation in routine diagnostic laboratories.

Idiotype (Id) vaccination provides an interesting immunotherapeutic strategy against B-cell lymphomas. In multiple myeloma (MM), however, the therapeutic efficacy of Id vaccination has been disappointing. In an attempt to improve the antitumoral potential, we added granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 2 (IL-2) to the protocol. Balb/c mice were inoculated i.p. (d 2) with different doses (1-5 x 10(5)) of HOPC myeloma cells secreting the Ig(HOPC) Id protein. Two days later, animals were injected with 10,000 U GM-CSF i.p. for 6 d consecutively (d 0-5). On d 5 and 11, myeloma-specific immunoglobulin (Ig(HOPC)) was administered i.p. together with incomplete Freund adjuvans followed by IL-2 (2 x 10,000 U/d; i.p) for 10 d (d 5-14). In animals inoculated with 10(5) myeloma cells, treatment with IL-2 given as a single agent prolonged the median survival time (MST, 67 d) when compared with the tumour control group (MST 48 d), whereas GM-CSF did not elicit any survival benefit (MST 49 d). Complete tumour rejection could be achieved in 27% (4/15) by the combination of Id vaccination and GM-CSF. Additional treatment with IL-2 further increased antimyeloma activity. In this case, 59% of the animals showed no signs of tumour recurrence. In mice with high tumour burden (5 x 10(5)), no treatment modality achieved long-term survivors. Both natural killer (NK) cells and CD8+ T cells may be involved in the anti-tumoural immune response. These data provide evidence for the combined use of GM-CSF and IL-2 to enhance the therapeutic effectiveness of clinical cancer vaccination protocols.

Idiotype (Id) vaccination provides an innovative treatment modality against B-cell malignancies. In multiple myeloma patients, however, the antitumoral potential of this immunotherapeutic concept is limited. In an attempt to improve the therapeutic effectiveness of Id vaccination, we added Flt3 ligand (Flt3-L) and interleukin 2 (IL-2) to the protocol. Balb/c mice were inoculated i.p. (d -2) with different doses (1-5 x 10(5)) of HOPC myeloma cells, secreting the IgHOPC Id-protein. Two days later, animals were treated with Flt3-L (10 microg per mouse/d, given i.p) for 10 consecutive days (d 0-9). On d 5 and d 11, myeloma-specific immunoglobulin (Ig(HOPC)) was administered s.c., together with incomplete Freund adjuvans (IFA) followed by the administration of IL-2 (2 x 10.000/d given i.p) for 10 d (d 5-14). Whereas Ig(HOPC), Flt3-L or IL-2, given alone, did not elicit long-term survival, the combination of IL-2 or Flt3-L with Id vaccination achieved a complete tumour rejection in 27% and 41% of mice respectively. However, the most powerful antimyeloma effects were induced by Flt3-L + Id vaccination + IL-2: 81% of the treated animals experienced long-term survival (> 180 d). Both natural killer (NK) cells and CD8+ T cells may be involved in the antitumoral immune response. These data suggest that the combination of Flt3-L and IL-2 can be used to enhance the therapeutic effectiveness of clinical cancer vaccination protocols.