The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

Monoclonal gammopathy of uncertain significance (MGUS) and smoldering multiple myeloma (SMM) are plasma cell disorders with a risk of progression of approximately 1% and 10% per year, respectively. We have previously shown that the proportion of bone marrow (BM) aberrant plasma cells (aPCs) within the BMPC compartment (aPC/BMPC) as assessed by flow cytometry (FC) contributes to differential diagnosis between MGUS and multiple myloma (MM). The goal of the present study was to investigate this parameter as a marker for risk of progression in MGUS (n = 407) and SMM (n = 93). Patients with a marked predominance of aPCs/BMPC (> or = 95%) at diagnosis displayed a significantly higher risk of progression both in MGUS and SMM (P< .001). Multivariate analysis for progression-free survival (PFS) selected the percentage aPC/BMPC (> or = 95%) as the most important independent variable, together with DNA aneuploidy and immunoparesis, for MGUS and SMM, respectively. Using these independent variables, we have identified 3 risk categories in MGUS (PFS at 5 years of 2%, 10%, and 46%, respectively; P< .001) and SMM patients (PFS at 5 years of 4%, 46%, and 72%, respectively; P < .001). Our results show that multiparameter FC evaluation of BMPC at diagnosis is a valuable tool that could help to individualize the follow-up strategy for MGUS and SMM patients.

Minimal residual disease (MRD) assessment is standard in many hematologic malignancies but is considered investigational in multiple myeloma (MM). We report a prospective analysis of the prognostic importance of MRD detection by multiparameter flow cytometry (MFC) in 295 newly diagnosed MM patients uniformly treated in the GEM2000 protocol VBMCP/VBAD induction plus autologous stem cell transplantation [ASCT]). MRD status by MFC was determined at day 100after ASCT. Progression-free survival (PFS; median 71 vs 37 months, P < .001) and overall survival (OS; median not reached vs 89 months, P ‫؍‬ .002) were longer in patients who were MRD negative versus MRD positive at day 100 after ASCT. Similar prognostic differentiation was seen in 147 patients who achieved immunofixation-negative complete response after ASCT. Moreover, MRD ؊ immunofixation-negative (IFx ؊ ) patients and MRD ؊ IFx ؉ patients had significantly longer PFS than MRD ؉ IFx ؊ patients. Multivariate analysis identified MRD status by MFC at day 100 after ASCT as the most important independent prognostic factor for PFS (HR ‫؍‬ 3.64, P ‫؍‬ . IntroductionIn most hematologic malignancies, response to front-line therapy is a good predictor of prognosis, with the longest survival seen in patients achieving an optimal response. This paradigm is represented by chronic myeloid leukemia (CML), in which hematologic, cytogenetic, and molecular remissions define progressively better response to therapy. In consequence, investigations to define these levels of remission are mandatory in routine clinical practice for treatment stratification and assessment of prognosis. 1 The situation is similar for other malignancies such as acute promyelocytic leukemia (APL) or acute lymphoblastic leukemia (ALL). 2,3 For this reason, there are continuous efforts to improve the sensitivity of the methods used to assess response to therapy, mainly through the introduction and refinement of both molecular and immunophenotyping approaches, as well as imaging techniques.Multiple myeloma (MM) should be no exception to this paradigm. For many years, the major goal of MM therapy was to achieve partial response (PR) or disease stabilization. 4,5 With the introduction of high-dose therapy plus autologous stem cell transplantation (HDT/ASCT), the new goal became the achievement of complete response (CR), defined as absence of M-protein by immunofixation (IFx) and less than 5% plasma cells (PCs) in bone marrow (BM). 6,7 More recently, the International Myeloma Working Group proposed a new response category of "stringent CR," which requires normalization of the free light chain ratio and the absence of residual clonal cells in the BM by immunohistochemistry or immunofluorescence. 8 As noted previously, the assessment of minimal residual disease (MRD), residual tumor cells persisting after therapy, is part of the standard of care in many hematologic malignancies, whereas in MM this is still considered investigational. Thus, MRD studies in MM have involved mainly small series of patients or have...

Multiple myeloma represents an incurable disease, for which development of new therapies is required. Here, we report the effect on myeloma cells of LBH589, a new hydroxamic acidderived histone deacetylase inhibitor. LBH589 was a potent antimyeloma agent (IC 50 < 40 nmol/L) on both cell lines and fresh cells from multiple myeloma patients, including cells resistant to conventional chemotherapeutic agents. In addition, LBH589 potentiated the action of drugs, such as bortezomib, dexamethasone, or melphalan. Using gene array, quantitative PCR, and Western analyses, we observed that LBH589 affected a large number of genes involved in cell cycle and cell death pathways. LBH589 blocked cell cycle progression, and this was accompanied by p21, p53, and p57 upregulation. LBH589 induced cell death through an increase in the mitochondrial outer membrane permeability. LBH589 favored apoptosome formation by inducing cytochrome c release, Apaf-1 up-regulation, and caspase-9 cleavage. In addition, LBH589 stimulated a caspase-independent pathway through the release of AIF from the mitochondria. LBH589 down-regulated Bcl-2 and particularly Bcl-X. Moreover, overexpression of Bcl-X in multiple myeloma cells prevented LBH589-induced cell death. All these data indicate that LBH589 could be a useful drug for the treatment of multiple myeloma patients. (Cancer Res 2006; 66(11): 5781-9)

To the best of our knowledge, this is the first prospective analysis in which the prognostic impact of a relatively high number of antigenic markers has been simultaneously analyzed in a large series of uniformly treated patients, showing that the expression of several antigens (particularly CD28 and CD117) on bone marrow plasma cells from patients with MM can help to identify patients at high risk of progression.

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Multiparametric immunophenotyping can be a sensitive method for analyzing the plasma cell (PC) compartment in patients with multiple myeloma because it discriminates between myelomatous and normal PCs. Using this approach, we compared the efficacy of high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) with that of conventional chemotherapy. We found that ASCT provided a significantly greater reduction in the level of residual tumor PCs and with better recovery of normal PCs. This profile of coexistence of normal PCs and myelomatous PCs resembled that observed in monoclonal gammopathy of undetermined significance. We also found that treatment-induced changes in the PC compartment correlated with disease outcome. Thus, patients in whom at least 30% of gated PCs had a normal phenotype after treatment had a significantly longer progression-free survival (60 ؎ 6 months versus 34 ؎ 12 months; P ‫؍‬ .02). (Blood. 2002;99: 1853-1856

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