Ralf Nowak

Shift of aberrant antigen expression at relapse or at treatment failure in acute leukemia.

The flow cytometric detection of aberrant antigen expression is one method proposed for the quantification of minimal residual disease (MRD) in acute leukemias. The present study was designed to investigate the stability of the aberrant antigen expression at relapse or at treatment failure of initial chemotherapy. For this purpose, multiparameter immunophenotyping with a panel of 15 monoclonal antibodies was used at diagnosis as well as at relapse (43 patients with overall 65 aberrations) and at treatment failure (35 patients with overall 66 aberrations). There was a significant decrease in the percentage of the initially described aberrant antigen expression on leukemia blasts at relapse (P = 0.001; n = 65) as well as at treatment failure (P = 0.0001; n = 66) considering all aberrations in the whole leukemia population. Concerning only patients with acute myelogenous leukemia (AML), significant decreases in the aberrant expression could be detected at relapse (P = 0.031; n = 42) and at treatment failure (P = 0.0001; n = 52). The changes in patients with acute lymphoblastic leukemia (ALL) were significant only at relapse (P = 0.006; n = 23). Initially, the most informative aberration was not detectable in four patients at relapse and in seven patients at treatment failure. A decrease of under 50% of the initial value was observed in another 8 patients at relapse and in 10 patients at treatment failure. In further studies assessing the detection of aberrant antigen expression for MRD, quantification of the relapses should be explicitly analyzed regarding the persistence of the initially described aberrant antigen expression.

Sensitivity of combined DNA/immunophenotype flow cytometry for the detection of low levels of aneuploid lymphoblastic leukemia cells in bone marrow.

By simultaneous DNA staining and immunophenotyping, the aneuploid blasts of acute lymphoblastic leukemia (ALL) can be quantitated by flow cytometry. The present study evaluates the application of this method for the detection of minimal residual disease (MRD). For this purpose leukemia cells with known aneuploidy were serially diluted with diploid bone marrow cells obtained after chemotherapy. These mixed samples were immunophenotyped using antibodies CD34, CD10, or CD19 and processed for DNA staining with propidium iodide. Fifty thousand cells were measured for each experiment and the results were compared with the analysis of control sample without added aneuploid cells. Aneuploid cells diluted to 1% were not detectable in 6/15 cases when DNA content analysis was used alone, and in dilutions of < 1%, these cells were not detectable. When DNA content and immunophenotype were simultaneously measured, aneuploidy was detectable in 15/15 cases at 1.00%, 6/6 at 0.50%, 16/16 at 0.10%, and 16/17 at 0.05%. Therefore this method may be useful for the detection of minimal residual disease in cases of aneuploid lymphoblastic leukemia.

A new PCR MIMIC strategy to quantify low mdr1 mRNA levels in drug resistant cell lines and AML blast samples

Determination of the MDR-phenotype in patients suffering from AML is an important hallmark of treatment outcome but is often complicated by technical problems in P-gp assessment. A PCR-MIMIC strategy was employed to construct PCR-fragments for a competitive and quantitative mdr1 reverse transcription-PCR-assay. Using K562 cells, which had been selected for drug resistance to the epipodophyllotoxin VP16, a stepwise increase of mdr1 levels depending on the concentration of VP16 was shown with the MIMIC technique. Comparison of mdr1 levels in drug selected K562 cells with the corresponding levels for P-gp and functional data indicated a mRNA threshold that has to be exceeded for the full expression of the MDR-phenotype. Subsequently mdr1 levels of 34 samples of de novo acute myeloid leukemia were determined with the PCR-MIMIC strategy. Ten patient samples could be identified with elevated mdr1 levels which were substantially lower than the levels observed in the MDR-cell line K 562 0.7 microM VP16. Outcome analysis revealed that eight of the ten patients had an unfavourable prognosis and did not achieve CR after induction chemotherapy. Coexpression of mdr1 and CD 34 was not associated with CR in all examined cases. Moreover all these patients had unfavourable cytogenetic aberrations. These data indicate a sensitive technique with applicability in patient material.

Detection of aneuploid cells in acute lymphoblastic leukemia with flow cytometry before and after therapy.

We have evaluated the proliferative activity and DNA content of immunophenotyped hematopoietic cells applying flow cytometry. After indirect immunofluorescence the cell membrane was permeabilized for propidium iodide staining of DNA. Compared with single parameter detection of DNA content this method has more certainty in the determination of aneuploidies in lymphatic leukemia cells. Immunophenotyped residual normal hematopoietic cells were used as an internal standard. If this method was tested for evaluation of therapeutic effects after chemotherapy greater sensitivity in detection of minimal residual disease was observed than when using microscopic evaluation or single parameter DNA analysis in cases of aneuploid lymphoblastic leukemias.

Complex cytogenetic and immunophenotypic aberrations in a patient with Sezary syndrome.

Sezary syndrome is defined as the leukemic variation of cutaneous T-cell lymphomas. Here we describe the cytogenetic pattern of peripheral T-cells of a 50-year-old male patient suffering from this disease. We used Giemsa-banding (G-banding) technique and a fluorescence in situ hybridization (FISH) assay to determine cytogenetic changes affecting 15 different chromosomes. The cells displayed an abnormal hypodiploid karyotype with a prominent insertion located at the short arm of chromosome 1. Unbalanced translocations were observed involving chromosomes 4 and 14. Besides other abnormalities we detected a 6q- deletion. These multiple genetic changes may reflect the high aggressivity of the neoplastically transformed T-cell population and the poor response to chemotherapeutic treatment.

Flow cytometric DNA-quantification of three-color immunophenotyped cells for subpopulation specific determination of aneuploidy and proliferation

A method is described for three-color immunophenotyping and simultaneous DNA-quantification using a flow cytometer equipped with a 488-nm argon laser and a mercury lamp (UV). The approach includes reproducible immunophenotyping comparing antigen expression before and after cell manipulation for DNA-measurement. The coefficients of variation after DNA-staining (CV=3.13 for T-cells in peripheral blood and CV=3.38 for T-cells in bone marrow) were adequate for exact DNA-analysis. For aneuploidy detection, a true internal standard was established measuring, for example, the DNA-content of T-cells in B-cell disease simultaneously with the DNA-content of the malignant cells. Using this method, aneuploidies could be unequivocally detected in 17 out of 24 patients with multiple myeloma. Furthermore, intratumor heterogeneities in DNA-content and antigen expression could be recognized, allowing an exact separation of tumor cells and normal hematopoiesis. The study also demonstrated the importance of exact immunophenotypic characterization of lymphocyte subpopulations and the determination of their specific proliferation, for example after proliferation induction in cell cultures. Future studies should address the applicability of this rather simple multiparameter approach for simultaneous immunophenotyping and DNA-measurement especially in the detection of minimal amounts of aneuploid cells after chemotherapy.

Flowcytometric Detection of Aneuploidies in Acute Lymphoblastic Leukemia (ALL) Before and After Chemotherapy

We performed flowcytometric determination of DNA content in immunophenotyped ALL cells. With this method one can increase the certainty of detecting aneuploidies compared with the commonly used one-parameter DNA analysis. We had found aneuploidies in 37% (9 of 24) of ALL patients. The proposed method can be used for detecting residual aneuploid leukemia cells after chemotherapy. Therefore we carried out experiments in which we mixed diploid bone marrow cells with the leukemic aneuploid cells for evaluating the sensitivity of our method. The results show that the sensitivity of the described flowcytometric method is much higher than those of the microscopical evaluation to look for residual leukemic cells. Important factors which influence the aneuploidy detection are the DNA index of the aneuploid population and the coefficient of variation of the G0/G1 peak of the cells. It should be further investigated whether this method can be used in monitoring patients with aneuploid leukemia cells.

Comparison of Growth Patterns of Acute Myeloid Leukemia Cells In Vitro and In Vivo by Flow Cytometry

Cells from patients with acute myeloid leukemia were analyzed by flow cytometric detection of PI staining for evaluation of proliferative activity. The distribution in cell cycle was measured immediately after bone marrow aspiration (as in vivo S-phase) and after 72 h cultivation without and with interleukin 3 (IL 3). As expected, in most cases (20/38) proliferation of blasts was stimulated with IL 3. But in four leukemias an increase was observed in control culture compared with in vivo S-phase. Interestingly, most cases of FAB M4 and M5 (9/13) were not sensitive to IL 3, but inversely most cases of FAB Ml and M2 showed a proliferation induction with IL 3 (13/18).