The World Health Organization (WHO) classification contributes to refined classification and prognostication of myelodysplastic syndromes (MDSs). Flow cytometry might add significantly to diagnostic and prognostic criteria. Our analysis of bone marrow samples from 50 patients with MDS showed aberrant expression of differentiation antigens in the myelomonocytic lineage. This also accounted for refractory anemia (RA) with or without ringed sideroblasts (RS), indicating multilineage dysplasia. In 38% of patients, CD34 ؉ myeloid blasts expressed CD5, CD7, or CD56. Flow cytometry data were translated into a numerical MDS flow-score. Flow-scores increased significantly from RA with or without RS, refractory cytopenia with multilineage dysplasia (RCMD) with or without RS up to refractory anemia with excess of blasts-1 (RAEB-1) and RAEB-2. No significant differences were observed between WHO cytogenetic subgroups. Flow-scores were highly heterogeneous within International Prognostic Scoring System (IPSS) subgroups. Patients in progression to advanced MDS or acute myeloid leukemia had a significantly higher flow-score compared with non-transfusion-dependent patients. In 60% of patients with transfusion dependency or progressive disease, myeloid blasts expressed CD7 or CD56, in contrast to only 9% of non-transfusiondependent patients. Moreover, all patients with pure RA with or without RS with aberrant myeloid blasts showed an adverse clinical course. In conclusion, flow cytometry in MDS identified aberrancies in the myelomonocytic lineage not otherwise determined by cytomorphology. In addition, flow cytometry identified patients at risk for transfusion dependency and/or progressive disease independent of known risk groups, which might have impact on treatment decisions and the prognostic scoring system in the near future.

The myelodysplastic syndromes are a group of clonal hematopoietic stem cell diseases characterized by cytopenia(s), dysplasia in one or more cell lineages and increased risk of evolution to acute myeloid leukemia (AML). Recent advances in immunophenotyping of hematopoietic progenitor and maturing cells in dysplastic bone marrow point to a useful role for multiparameter flow cytometry (FCM) in the diagnosis and prognostication of myelodysplastic syndromes. In March 2008, representatives from 18 European institutes participated in a European LeukemiaNet (ELN) workshop held in Amsterdam as a first step towards standardization of FCM in myelodysplastic syndromes. Consensus was reached regarding standard methods for cell sampling, handling and processing. The group also defined minimal combinations of antibodies to analyze aberrant immunophenotypes and thus dysplasia. Examples are altered numbers of CD34 + precursors, aberrant expression of markers on myeloblasts, maturing myeloid cells, monocytes or erythroid precursors and the expression of lineage infidelity markers. When applied in practice, aberrant FCM patterns correlate well with morphology, the subclassification of myelodysplastic syndromes, and prognostic scoring systems. However, the group also concluded that despite strong evidence for an impact of FCM in myelodysplastic syndromes, further (prospective) validation of markers and immunophenotypic patterns are required against control patient groups as well as further standardization in multi-center studies. Standardization of FCM in myelodysplastic syndromes may thus contribute to improved diagnosis and prognostication of myelodysplastic syndromes in the future. Cancer and Immunology, Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. E-mail: a.vandeloosdrecht@vumc.nl © F e r r a t a S t o r t i F o u n d a t i o n

Purpose: In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event originates from the CD34 + CD38À stem cell compartment. Survival of these cells after chemotherapy may lead to minimal residual disease (MRD) and subsequently to relapse.Therefore, the prognostic impact of stem cell frequency in CD34-positive AML was investigated. Experimental Design: First, the leukemogenic potential of unpurified CD34 + CD38 À cells, present among other cells, was investigated in vivo using nonobese diabetic/severe combined immunodeficient mice transplantation experiments. Second, we analyzed whether the CD34 + CD38À compartment at diagnosis correlates with MRD frequency after chemotherapy and clinical outcome in 92 AML patients. Results: In vivo data showed that engraftment of AML blasts in nonobese diabetic/severe combined immunodeficient mice directly correlated with stem cell frequency of the graft. In patients, a high percentage of CD34 + CD38À stem cells at diagnosis significantly correlated with a high MRD frequency, especially after the third course of chemotherapy. Also, it directly correlated with poor survival. In contrast, total CD34 + percentage showed no such correlations. Conclusions: Both in vivo data, as well as the correlation studies, show that AML stem cell frequency at diagnosis offers a new prognostic factor. From our data, it is tempting to hypothesize that a large CD34 + CD38À population at diagnosis reflects a higher percentage of chemotherapyresistant cells that will lead to the outgrowth of MRD, thereby affecting clinical outcome. Ultimately, future therapies should be directed toward malignant stem cells.

Outgrowth of minimal residual disease (MRD) in acute myeloid leukaemia (AML) is responsible for the occurrence of relapses. MRD can be quantified by immunophenotyping on a flow cytometer using the expression of leukaemia-associated phenotypes. MRD was monitored in follow-up samples taken from bone marrow (BM) of 72 patients after three different cycles of chemotherapy and from autologous peripheral blood stem cell (PBSC) products. The MRD% in BM after the first cycle (n ¼ 51), second cycle (n ¼ 52) and third cycle (n ¼ 30), as well as in PBSC products (n ¼ 39) strongly correlated with relapse-free survival. At a cutoff level of 1% after the first cycle and median cutoff levels of 0.14% after the second, 0.11% after the third cycle and 0.13% for PBSC products, the relative risk of relapse was a factor 6.1, 3.4, 7.2 and 5.7, respectively, higher for patients in the high MRD group. Also, absolute MRD cell number/ml was highly predictive of the clinical outcome. After the treatment has ended, an increase of MRD% predicted forthcoming relapses, with MRD assessment intervals of p3 months. In conclusion, MRD parameter assessment at different stages of disease is highly reliable in predicting survival and forthcoming relapses in AML. Leukemia (2004) IntroductionMinimal residual disease (MRD) cells, present in the bone marrow (BM) of patients with acute myeloid leukaemia (AML) after chemotherapy, are thought to be responsible for the emergence of a relapse. Monitoring of MRD cells by molecular biologic or immunophenotypic detection methods are thought to be of crucial value for defining individualized treatment of patients. Based on MRD frequencies patient-tailored postremission chemotherapy could be designed, which might reduce either the risk of relapse or, in case of withholding therapy to the patient, diminish morbidity and mortality. Also, when impending relapses can be predicted, these might be prevented by early therapeutic interventions. Furthermore, MRD frequency assessment can be used to quantify the leukaemia cell contamination of an autologous peripheral blood stem cell (PBSC) product in order to guide the decision of whether or not to purge, and in the latter case to establish the efficacy of leukaemia cell eradication.Immunophenotypic detection of MRD by flow cytometry can be performed by defining aberrant marker expressions, the socalled leukaemia-associated phenotypes (LAPs), on malignant cells at diagnosis. 1-6 These LAPs are not, or only in very low frequencies, present on normal blood or BM cells. This technique can be successfully applied in up to 80% of the AML patients in contrast to molecular biological techniques, which were until now applicable only in 20-30% of the cases. 7-9 So far, in AML the predictive value of MRD frequency detection using immunophenotyping has been reported by a few groups only. 6,[10][11][12] In adult AML patients, San Miguel et al 6,10 demonstrated the prognostic value of MRD after induction and intensification therapy, while Venditti et al 11 have found this after con...