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B-cell chronic lymphocytic leukaemia (B-CLL) results from the clonal expansion of mature B lymphocytes. The detection of leukaemia-associated genetic markers in CD34-positive progenitor cells in a subset of B-CLL patients suggests that malignant transformation in B-CLL occurs in an immature progenitor cell compartment. To further quantify the percentage of B-CLL patients with genetically aberrant progenitor cells we have investigated CD34+ bone marrow cells in 11 B-CLL patients at the single cell level by simultaneous genetic and immunophenotypic analysis (FICTION). In five patients with trisomy 12, CD34+ haemopoietic progenitor cells were detectable on bone marrow smears. In one patient with trisomy 12, CD34+ progenitor cells were isolated by FACS sorting. In all six patients trisomy 12 was not found in the CD34+ cells. Progenitor cells were also analysed in three patients with Rb-deletion and in two patients with deletion of p53. In all patients the genetic marker was not detected in the CD34+ cells. In conclusion, we did not find genetically aberrant progenitor cells in this group of B-CLL patients. These results suggest that the subset of B-CLL patients with genetically aberrant CD34+ cells may be very small. This is of significance for our understanding of B-CLL biology and for future strategies using autologous stem cell transplantation.

Analysis of differentiation antigens on leukaemic blasts is routinely done for diagnostic purposes, i.e. determination of stage of differentiation and lineage assignment. Acute lymphoblastic leukaemias are also frequently characterized by a leukaemia-associated immunophenotype (LAIP), either the coexpression of differentiation antigens physiologically restricted to other stages of differentiation (asynchronous LAIP) or cell lineages (aberrant LAIP). We defined LAIP in 241 consecutive unselected B-lineage (n = 193) and T-lineage (n = 48) ALL by three-colour flow cytometry using directly conjugated monoclonal antibodies. The incidence of LAIP was found to be 91.7%. In 63% of patients two to six leukaemia-associated expression patterns were detected. In order to study the specificity of LAIP in a therapy-relevant setting, remission bone marrow samples from patients with B-lineage ALL were analysed for the expression of T-lineage-associated phenotypes on the normal bone marrow cells and vice versa. The frequency of all T-lineage LAIP+ cells and all aberrant B-lineage LAIP+ cells was1% in regenerating bone marrow samples at different timepoints. The incidence and clinical significance of LAIP+ cells was studied in 196 remission marrows of 70 ALL patients (55 remaining in CCR, 14 with bone marrow relapse, one with isolated CNS relapse). The presence of1% LAIP+ at two consecutive timepoints predicted 5/8 bone marrow relapses in B-lineage ALL. The occurrence of LAIP+ cells1% in T-lineage ALL after induction therapy predicted relapse in 7/7 cases. In conclusion, flow cytometric detection of LAIP+ cells appears to be a powerful tool for the prediction of outcome in ALL.

Quantitative determination of hematopoietic progenitor cells is a major issue in peripheral blood progenitor and stem cell collection and transfusion, although the extent is still an object of discussion.In 116 leukapheresis collections from 42 patients, immunophenotyping for CD34+ cells, evaluation of in vitro proliferative capacity by a colony-forming unit-granulocyte-macrophage (CFU-GM) assay, and viability assessment by trypan blue exclusion were performed before and after storage in liquid nitrogen at -196 degrees C.Before storage, the median number of CD34+ cells was 1.46 x 10(6) (range, 0.01-54.05 x 10(6)) per kg of body weight (BW). There was no significant difference between precryopreservation and postcryopreservation numbers. The median number of CFU-GM was 2.25 x 10(5) (range, 0.02-157.49 x 10(5)) per kg of BW before cryopreservation and significantly (p0.001) lower, 0.83 x 10(5) (range, 0-220.36 x 10(5)) per kg of BW, after cryopreservation. The correlation coefficient of prestorage and poststorage values was 0.92. The median ratio of poststorage and prestorage values was 42.3 percent (0-304.8%). Male patients who underwent intense chemotherapy (5 cycles) showed a significantly lower ratio of postcryopreservation and precryopreservation CFU-GM values than other patients (p = 0.0047). A strong linear correlation was determined between the number of CD34+ cells per kg of BW and the number of CFU-GM per kg of BW before and after cryopreservation. A viability below 50 percent predicted a high loss of in vitro proliferative capacity, while a viability above 50 percent did not correlate with a high ratio of CFU-GM from after and before cryopreservation.A good correlation between the variables used for characterization of peripheral blood progenitor cells--the number of CD34+ cells and the number of CFU-GM--was observed. Viability assessment by trypan blue exclusion does not seem to be a substitute for assays evaluating in vitro proliferative capacity.

B-cell chronic lymphocytic leukemia (B-CLL) is a slowly progressive disease characterized by the clonal expansion of CD5+/CD23+ B lymphocytes. The malignant transformation is assumed to occur at the level of mature B lymphocytes. We asked whether CD34+ progenitor cells are involved in the malignant process in B-CLL. Furthermore, we investigated the possibility of aberrant CD34 expression by the malignant B-cell clone. Bone marrow and peripheral blood samples from 75 patients with B-CLL were tested for the presence of trisomy 12 and deletion of the retinoblastoma gene (Rb) by fluorescence in situ hybridization. CD34+ subpopulations were isolated by fluorescence-activated cell sorting and analyzed for the presence of the informative genetic marker. Bone marrow and peripheral blood samples of 10 B-CLL patients were analyzed for coexpression of CD34/CD5/CD20. Trisomy 12 was detected in 15 of 75 (20%) and Rb-deletion was detected in 6 of 30 patients (20%). In 7 patients with trisomy 12, hematopoietic progenitor cells were sorted, with the sort purity being between 85% and 99.8%. The genetic marker was detected in the CD34+/CD38+ cells as well as in the CD34+/38− subsets in 3 patients. Progenitor cells were also sorted in 2 patients with Rb-deletion. In 1 patient, Rb-deletion was present in 10% of CD34+/38+ cells. In the other patient, Rb-deletion was neither detected in the CD34+/38+ nor in the CD34+/CD38− subsets. In all 10 patients investigated for coexpression of CD34/CD5/CD20, we could not find a subpopulation coexpressing these markers. We conclude that trisomy 12 and Rb-deletion are present in a considerable subset of patients with B-CLL. In part of these patients, the genetic marker was detected at the level of CD34+ stem cells. CD34 expression is not related to an aberrant phenotype of the malignant B-cell clone. These results suggest that the malignant transformation in B-CLL may involve early hematopoietic stem cells and place a note of caution on future strategies using autologous stem cell transplantation.

It has been supposed in de novo AML that malignant transformation occurs at the level of committed progenitors. Recent data of our group and others provide evidence that in AML malignant transformation may regularly occur at the level of stem cells. These cells can be discriminated by function and specific surface molecules. CD34, a glycophosphoprotein, is a cellular surface antigen characteristically expressed by stem cells. CD34+ stem cells can be further subdivided by the expression of additional surface molecules like CD38 and CD117. In this article we present results from cytogenetic examinations of FACS-isolated stem cell subpopulations in eight patients (four AML and four MDS). Six of them displayed clonal karyotype abnormalities at the time of first diagnoses in the native bone marrow (5q-; 5q- and complex abnormalities; +8; inv(16) and +8; i(17q) and -21; i(21q)). We used CD117, the receptor for the stem cell factor (also KIT oncogene) as a new cellular surface marker. CD34+/CD117+/- stem cell subpopulations were examined in two patients with AML and three patients with MDS. We found leukemic stem cells in every type of stem cell subpopulation examined (CD34+/CD38-, CD34+/CD38+, CD34+/CD117-, CD34+/CD117+). Secondary, progression-associated chromosome abnormalities likewise were demonstrable in CD34+ cells. In three patients a mosaic of normal and abnormal metaphases was found in the highly purified stem cell subpopulations. We conclude that in AML and MDS stem cells are the target of leukemogenic genetic defects. CD117 as a new marker to isolate different CD34+ subpopulations was not sufficient to discriminate between normal and leukemic stem cells. Our findings have implications for autologous stem cell transplantation, high-dose chemotherapy and the pathogenetic concept of leukemogenesis.

Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38-(early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38-populations and in 9 of the CD34+/CD38-specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38-cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38-cells with multilineage potential.

B-cell chronic lymphocytic leukemia (B-CLL) is a slowly progressive disease resulting from the clonal expansion of mature B lymphocytes. The most frequent chromosomal abnormality is trisomy 12. Recently more aggressive therapeutic approaches using myeloablative therapy and autologous stem-cell support have been developed. Phase I/II studies have resulted in molecular remission and prolonged survival. One cause of relapse may be tumor-cell contamination of the transplant. We asked whether immunophenotypically identified hematopoietic progenitor cells are part of the malignant cell population in B-CLL. In a patient with trisomy 12, two subpopulations of hematopoietic progenitor cells, CD34+/CD38+ and CD34+/CD38- cells, were isolated by fluorescence-activated-cell-sorting; the sort purity was 98%. Trisomy 12 was detected in 13% of CD34+/38+ cells and in 34% of CD34+/38- cells. These data suggest that CD34+ cells are involved in the malignant clone of patients with B-CLL. The results are of significance for future strategies using autologous stem-cell transfusion.

In this study we addressed the question whether hematopoietic stem cells characterized by the expression of CD34 are involved in the leukemogenic process in AML and MDS. For this purpose bone marrow cells were sorted according to their expression of CD34 and coexpression of CD38 or CD117 (stem cell factor receptor = CKIT). The sorted subpopulations were genetically analyzed either by cytogenetics or FISH. Successful cytogenetics of sorted CD34+ subpopulations could be performed in 24/54 pts. included in our study (AML: n = 18, MDS: n = 6). At diagnosis 18 displayed a wide variety of different clonal karyotype abnormalities in the unsorted bone marrow: add(2q); +4; 5q−; 5q− and complex anomalies; t(6;9); 6p−; −7; +8; t(8;21); −12; inv(16); i(17q); t(17;20); −21; i(21q). We analyzed sorted stem cell subpopulations with the immunophenotype CD34+/CD38± in 19 pts., in 5 pts. stem cells with the immunophenotype CD34+/ CD117± were examined. We found genetically abnormal stem cells in every subpopulation (CD34+/CD 38−, CD34+/CD38+, CD34+/CD117−, CD34+/CD117−) in every informative case (abnormal karyotype in the unsorted bone marrow and metaphases/interphase cells available for cytogenetic analysis). In 11 pts. a mosaic of normal and abnormal cells was observed in the sorted stem cell subpopulations. In five pts. secondary, progression-associated anomalies were present in the sorted stem cells. We conclude that in AML and MDS malignant transformation and disease progression occur at the level of immature hematopoietic stem cells independent from the phenotype of the leukemic bulk population and the type of genetic alteration.

The phenotype of the (pre)leukemic stem cell in AML and MDS has not been identified as yet. We have investigated whether cells with a stem cell-like immunophenotype (CD34+/CD38-) are involved in the leukemogenic process. In 12 patients with AML and in one patient with MDS the identification of (pre)leukemic cells was performed by classical cytogenetics. Highly purified (96-98% purity) cellular subpopulations were generated by fluoresence activated cell sorting according to the expression of CD34 and CD38. Following brief incubation with a cytokine cocktail (EPO, GCSF, GM-CSF, IL-3, SCF) the sorted subpopulations were processed for chromosome analysis. In 9/12 AML patients clonal karyotype abnormalities were observed in the unsorted material. In 7/9 cases the chromosomal changes found in unsorted specimens were also detected in the immature stem cell-like population (CD34+/CD38-). In 9/9 cases the karyotype abnormalities were diagnosed in the population of committed progenitors (CD34+/CD38+). Additional secondary abnormalities were also demonstrable in both subpopulations in a case of M4Eo with inv(16). In a patient with secondary MDS RA, 2 subpopulations, CD34+/CD38- and CD34+/CD38+, were sorted together (CD34+/ 38+ -) because of the rarity of available cells. Cytogenetic analysis revealed a mosaic of normal and abnormal cells with 5 different dependent cell clones. All abnormal cell clones were demonstrable in the CD34+, sorted cell population. The relevance of our findings for pathogenesis, autologous stem cell transplantation and targeted gene therapy are discussed.

B-cell chronic lymphocytic leukemia (B-CLL) is a slowly progressive disease resulting from the clonal expansion of mature B lymphocytes. The most frequent chromosomal abnormality is trisomy 12. Recently more aggressive therapeutic approaches using myeloablative therapy and autologous stem-cell support have been developed. Phase I/II studies have resulted in molecular remission and prolonged survival. One cause of relapse may be tumor-cell contamination of the transplant. We asked whether immunophenotypically identified hematopoietic progenitor cells are part of the malignant cell population in B-CLL. In a patient with trisomy 12, two subpopulations of hematopoietic progenitor cells, CD34+/CD38+ and CD34+/CD38- cells, were isolated by fluorescence-activated-cell-sorting; the sort purity was 98%. Trisomy 12 was detected in 13% of CD34+/38+ cells and in 34% of CD34+/38- cells. These data suggest that CD34+ cells are involved in the malignant clone of patients with B-CLL. The results are of significance for future strategies using autologous stem-cell transfusion.

Tumour cells from a patient with recurrent testicular germ cell cancer and bone marrow infiltration were found to express CD33 and CD34 in the absence of other haemopoiesis-associated antigens. After myelosuppression and treatment with G-CSF for stem cell mobilization, CD34-positive tumour cells were detected in the peripheral blood in addition to normal haemopoietic progenitor cells. The tumour cells were decreased in the leukapheresis product. Retrospectively, the appearance of tumour cells in the peripheral blood after stem cell mobilization was the first indication of impending relapse.

CD34 positive progenitor cells were analyzed in the bone marrow aspirate from a patient with newly diagnosed AML FAB M4. The patient had trisomy 4 as sole cytogenetic abnormality and a dominant population of CD34 negative leukemic blasts. Karyotyping of the FACS isolated, minor subpopulation of CD34+/CD38-, 'stem cell'-like cells (incidence 0.29%) revealed trisomy 4 in 11/13 metaphases. No metaphases were obtained in the CD34 negative subpopulation. The experiments point to the existence of leukemic stem cells in the CD34+/CD38- compartment in AML patients with trisomy 4.