MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia.

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=51), second cycle (n=52) and third cycle (n=30), as well as in PBSC products (n=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 < or =3 months. In conclusion, MRD parameter assessment at different stages of disease is highly reliable in predicting survival and forthcoming relapses in AML.

A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia.

Minimal residual disease (MRD) cells are thought to be responsible for the persistence and relapse of acute myeloid leukemia (AML). Flow cytometric MRD detection by the establishment of a leukemia-associated phenotype (LAP) at diagnosis can be used in 80% of AML patients, allowing detection and functional characterization of MRD in follow-up bone marrow. One of the mechanisms contributing to inefficient chemotherapy is apoptosis resistance. Measuring apoptosis parameters in MRD cells will help to unravel the importance of apoptosis resistance in AML. We therefore developed a four-color flow cytometry method that enables establishment of apoptosis-related protein expression such as Bcl-2, Bcl-x(L), Mcl-1 and Bax at diagnosis and in MRD. Firstly, validation of this assay using Western blot analysis in five leukemia cell lines showed a significant correlation (R=0.70: P<0.0001). Secondly, the influence of the permeabilization procedure on LAP expression was investigated in 38 AML samples at diagnosis and in 42 MRD samples. Quantification of the frequency of LAP+ cells with and without permeabilization showed no significant differences (diagnosis: P= 0.57, follow-up: P= 0.43). The flow cytometric protocol thus enables analysis of apoptosis-related proteins at different stages of the disease, which will lead to a better understanding of the role of apoptosis resistance in the emergence of MRD in AML.

High percentage of CD34-positive cells in autologous AML peripheral blood stem cell products reflects inadequate in vivo purging and low chemotherapeutic toxicity in a subgroup of patients with poor clinical outcome.

In this study, a high CD34% in autologous peripheral blood stem cell (PBSC) products from 71 AML patients was associated directly with a high relapse rate (P = 0.006) and inversely with disease-free survival (P = 0.003), irrespective whether patients were transplanted or not. The relapse rate at 12 months was 67% in a group with >0.8% CD34+ cells and 34% in a group with < or = 0.8% CD34+ cells. Although the percentage of malignant CD34+ cells in the CD34+ compartment in the relapses of the first group was not high (median 8%), the total number of malignant cells as a percentage of WBC was about 13 times higher than for the patients remaining >12 months in remission. When all patients evaluable were taken together, this frequency of malignant cells correlated strongly with disease-free survival (P < 0.001). Both this massive mobilization of normal CD34+ cells and high frequency of malignant cells in the subgroup of patients with CD34 >0.8% and relapse within 12 months indicate an insufficient in vivo purging, as well as low chemotherapeutic bone marrow toxicity. This was confirmed by an inverse correlation between hypoplasia period after the induction therapy and CD34% in PBSC products (P < 0.002). It is concluded that a subgroup of patients has been identified that might benefit from a more intensive chemotherapeutic treatment.

Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity in acute myeloid leukemia.

Relapse is common in acute myeloid leukemia (AML) due to persistence of residual leukemia cells: minimal residual disease (MRD). In 102 out of 127 patients (80%), cells at diagnosis displayed one or more leukemia-associated phenotypes (LAP), ie combinations of cell surface markers which are absent in normal cells and can thus be used to detect MRD at follow-up. Functional characterization of MRD cells for P-glycoprotein (Pgp) and multidrug resistance protein (MRP) activity is essential to investigate the role of these drug transport proteins in multidrug resistance in AML. A fluorescent probe assay using Syto16/PSC833 and calcein-AM/probenecid as substrate/modulator of the Pgp and MRP pump, respectively, and subsequent labeling of cells with monoclonal antibodies for LAP detection allowed simultaneous detection of LAP and Pgp or MRP activity. Validation of this assay is shown for 30 newly diagnosed AML and 11 MRD situations. In addition, no significant differences were found when comparing fresh and cryopreserved de novo AML for LAP expression (n = 43), Pgp (n = 30) and MRP (n = 24) function and for MRD samples for simultaneous LAP expression and Pgp/MRP activity (n = 10). This approach enables longitudinal and multicenter studies on the detection, quantification and functional characterisation of MRD cells.

Isolation of the intact white pulp. Quantitative and qualitative analysis of the cellular composition of the splenic compartments.

The spleen is anatomically and functionally divided into two compartments: the red pulp, where particles are effectively removed from the blood, and the white pulp, where specific immune responses are generated. Here the isolation of white pulp from red pulp is described, allowing a detailed analysis of the cellular components of both red and white pulp separately. A striking abundance of memory T cells was found in the white and red pulp with an overall ratio of T and B cells in the white pulp being similar to that in lymph nodes. Both NK and gamma delta T cells can be found in white pulp and lymph nodes, but granulocytes are absent. The distribution of dendritic cell subsets showed significant differences between white pulp and lymph nodes. Furthermore, short-term homing experiments showed that migration of lymphocytes into the white pulp greatly exceeded that into lymph nodes, with significant differences in migration of various lymphocytes subsets. This suggests a different migration and retention mechanism in the white pulp. This new isolation technique will allow further analysis of the functional capacities of the splenic compartments.