title: Multiparametric analysis of apoptotic and multi-drug resistance phenotypes according to the blast cell maturation stage in elderly patients with acute myeloid leukemia.
abstract: OBJECTIVE
  Acute myeloid leukemia (AML) is a heterogeneous group of malignant diseases, often characterized by coexistence of more than one subpopulation of blast cells. Multiparametric flow cytometry immunophenotyping has proven to be a reliable and sensitive approach for the discrimination of myeloid blast cells from residual normal cells present in bone marrow samples from AML patients and, at the same time, allows the identification of different maturation compartments among myeloid blasts. Therefore, it provides a unique tool for assessing apoptotic and multidrug resistance (MDR)-associated phenotypes in individual subsets of leukemic cells.
  
  
  METHODS
  The aim of the present study was to explore the simultaneous expression of proteins related to both apoptosis (APO2.7, bcl-2, bax) and multidrug resistance (MDR1, MRP, LRP) in the different blast cell subpopulations detected at diagnosis in a group of 72 elderly patients with AML. In addition, we included 5 bone marrow samples from healthy adult donors in the analysis.
  
  
  RESULTS
  Immature blast cells (CD34+: subset I) showed a significantly higher level of bcl-2 expression (p <0.0001) together with a lower reactivity for APO 2.7 (p=0.02) as compared to the other more mature CD34- cell subsets. The expression of Bax parallelled that of APO 2.7, although the difference between immature CD34+ blast cells and the mature blast cell subsets did not reach statistical significance (p=0.18). These results translated into a significantly (p<0.0001) higher bcl-2/bax ratio for the CD34+ blast cells as compared to that of the two CD34- blast cell subpopulations. Regarding the expression of the multidrug resistance-associated proteins Pgp and MRP, CD34+ blast cells displayed a greater expression of both proteins as compared to the more mature CD34- AML blast cells, but differences according to maturation stage of AML blast cells did not reach statistical significance. In contrast, LRP expression was significantly lower in the more immature CD34+ blast cell subset than in the more mature ones (p=0.01).
  
  
  CONCLUSIONS
  As far as normal bone marrow is concerned our results suggest that all blast cell subpopulations are more protected from apoptosis than their normal counterparts. We conclude that in elderly patients with AML the more immature blast cells are more resistant to apoptotic processes, which could explain why, when AML relapses, the blast cells frequently display a more immature phenotype than that observed at diagnosis. Contradictory results in multidrug resistance profile support the hypothesis that failure to respond to chemotherapeutic drugs in AML is a multifactorial phenomenon.
authors.affiliation: Department of Hematology, University Hospital of Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Suárez
authors.forename: B
authors.initials: B
authors.lastname: Vidriales
authors.forename: J
authors.initials: J
authors.lastname: García-Laraña
authors.forename: A
authors.initials: A
authors.lastname: López
authors.forename: R
authors.initials: R
authors.lastname: Martínez
authors.forename: V
authors.initials: V
authors.lastname: Martín-Reina
authors.forename: M
authors.initials: M
authors.lastname: Tormo
authors.forename: J D
authors.initials: JD
authors.lastname: González-San Miguel
authors.forename: E
authors.initials: E
authors.lastname: Lavilla
authors.forename: R
authors.initials: R
authors.lastname: García-Boyero
authors.forename: A
authors.initials: A
authors.lastname: Orfao
authors.forename: J F
authors.initials: JF
authors.lastname: San Miguel
mesh_terms: D000208:Acute Disease; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis / Q000235:genetics*; D001752:Blast Crisis / Q000473:pathology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D015999:Multivariate Analysis; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology; D010641:Phenotype