Detection of Cytogenetic Aberrations Both in CD90 (Thy-1)-Positive And
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Leukemia (1999) 13, 1770–1775 1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu Detection of cytogenetic aberrations both in CD90 (Thy-1)-positive and (Thy-1)- negative stem cell (CD34) subfractions of patients with acute and chronic myeloid leukemias C Brendel1, B Mohr1, C Schimmelpfennig1,JMu¨ller1, M Bornha¨user1, M Schmidt1, M Ritter1, G Ehninger1 and A Neubauer2 1Universita¨tsklinikum Carl Gustav Carus, Medizinische Klinik I, Dresden; and 2Universita¨tsklinikum der Philipps-Universita¨t Marburg, Abt. fu¨r Ha¨matologie, Onkologie und Immunologie, Zentrum fu¨r Innere Medizin, Marburg, Germany Acute myeloid leukemia (AML) and chronic myeloid leukemia early stem cells from malignant progenitor cells via distinct (CML) are thought to arise from malignant hematopoietic pro- surface marker molecules is a challenging perspective, since genitor cells representing early and undifferentiated stem cell various improved cell selection methods have been recently clones. In CML there is evidence for a progenitor cell subset 4 free of leukemic clones, depending on the course of the dis- applied. ease. Additionally, it has been suggested that in AML, the early Acute myeloid leukemia is also considered to be an early stem cell compartment (CD34+/90+) does not harbor the malig- stem cell disease with fulminant lethal course if untreated. nant clone. We analyzed white blood cells from leukemia Since only a minority of patients achieve long-term complete patients for the presence of aberrant cells in stem cell subfrac- remission, bone marrow or stem cell transplantation is still the tions. Sixteen patients with CML, six patients with AML, two patients with acute lymphatic leukemia (ALL) and one with treatment of choice if appropriate donor cells are available. chronic myelomonocytic leukemia (CMMOL), all with known Specific chromosomal abnormalities can be determined in cytogenetic abnormalities, were evaluated according to their some FAB subtypes. Furthermore, various gross chromosomal CD90 (Thy-1)-positive or -negative phenotype. Subsets were aberrations can be discovered in 60% de novo AML of differ- sorted on to slides and further characterized by FISH and/or ent subtypes; in secondary AML multiple aberrations occur standard cytogenetic testing. The bcr-abl translocation or even more frequently.5 Therefore, structural chromosome gross chromosomal abnormalities could be detected in equally high amounts of 92.2% and 89.2% in both stem cell subsets. abnormalities can often serve as distinct markers for the malig- We conclude, that in progressed AML and CML cells charac- nant cell clone. Interestingly, CD34-positive/CD90-positive or terized by specific genetic aberrations implicated in the malig- CD34/CD90-negative hematopoietic progenitor cells from nant state can be found in the CD34+/CD90+ and CD90− popu- patients with AML seem to exhibit different long-term prolifer- lation, thus making CD90 an inappropriate marker to ative abilities and different cytogenetic characteristics with distinguish benign from malignant cells in these leukemias. regard to the leukemic clone.6 These recent data offer new Keywords: hematopoietic progenitor cells; CD90; CD34; leukemia; FISH; cell sorting aspects for obtaining purged stem cell grafts for autologous transplantation in both diseases. Early hematopoietic progenitor cells are characterized through their undifferentiated proliferative potential and their Introduction expression of CD34 surface antigen. HLA-DR, CD38, c-kit, IL6-receptor and CD90 are also associated with hematopoietic Chronic myeloid leukemia is characterized through the progenitor cells, but not exclusively. The CD90 surface anti- expansion of an early stem cell clone mostly of myeloid origin gen is also expressed on human B and T lymphocytes and harboring the Philadelphia (Ph) chromosome. At the molecu- neural cells.7,8 Cells coexpressing the CD34 and CD90 anti- lar level this typical cytogenetic finding represents the translo- gens are thought to be a stem cell subset that contains a high cation t(9;22)(q34;q11) with consecutive gene rearrangement number of long-term culture initiating cells.9,10 We sought to resulting in the chimeric bcr-abl fusion 210 kDa oncoprotein. investigate the occurrence of malignant clones in different The course of CML is usually fairly slow in the first phase of stem cell subsets of patients with CML and AML harboring the disease but will ultimately progress into an accelerated distinct chromosomal abnormalities. Cells sorted with a phase and an overt leukemic state, the blast crisis. Bone mar- + + fluorescence-activated cell sorter into CD34 /CD90 and row or peripheral stem cell transplantation from an HLA- + − CD34 /CD90 fractions were further analyzed cytogenetically identical sibling or from a matched unrelated donor is the only with FISH and/or standard cytogenetic techniques. We were chance for cure in these patients if they do not respond to unable to detect any cytogenetic difference in CD34-positive treatment with IFN-␣. If such a donor is not available autolog- progenitor cells of either CD90-positive or CD90-negative ous transplantation has been performed in some patients with phenotype. limited success because of residual leukemic cells in the graft. Different attempts have been made to obtain Ph chromosome- negative autologous graft cells.1,2 It has been shown pre- Materials and methods viously that stem cell subpopulations from patients with CML with either CD34-positive/DR-positive or DR-negative pheno- Patients type harbor bcr-abl rearrangements in a different manner, depending on the course of the disease.3 Differentiation of Blood samples were obtained from patients treated at the Universita¨tsklinikum Carl Gustav Carus in Dresden after infor- med consent. We examined blood from 13 patients with CML, Correspondence: A Neubauer, Universita¨tsklinikum der Philipps- two with primary diagnosis (PD), two in early chronic phase Universita¨t Marburg, Zentrum fu¨r Innere Medizin, Abt. Ha¨matologie, Onkologie und Immunologie, Baldinger Straβe, 35043 Marburg, (EAP), five in late chronic phase (LCP), five in accelerated Germany; Fax: ++49-6421-2866358 phase (AC) and three in blast crisis (BC). Additionally, blood Received 1 March 1999; accepted 24 June 1999 samples were taken from four patients with secondary AML, Cytogenetic aberrations in CD90 stem cell subfractions in AML and CML C Brendel et al 1771 one patient with de novo AML, one patient with first relapse regard to their CD90 phenotype as shown in Figure 1. In AML, two patients with ALL, one patient with CMMoL and selected cases purities of the sorted cells proved to be more two healthy donors (Table 1). Cells were either taken from the than 90% (range 90–98%). Cells were sorted on to slides and leukapheresis product or 20 ml peripheral blood were drawn into culture medium using a fluorescence-activated (FACS into heparinized collection tubes and submitted to Lympho- Vantage; Becton Dickinson) cell sorter. PBS (phosphate- prep (Nycomed, Oslo, Norway) gradient centrifugation buffered saline without Ca++ or Mg++) was used as sheath and according to standard protocols in order to separate a mono- sample fluid and cells were air dried in the PBS drop after nuclear cell fraction (MNC). sorting on to the slide. These stem cell subfractions were further submitted to FISH analysis and/or cytogenetic evaluation, respectively. Cell selection and sorting Depending on the amount of CD34-positive cells, MNC were Interphase-fluorescence in situ hybridization (I-FISH) either stained directly or further enriched for CD34 engaging the Isolex (Baxter, Unterschleissheim, Germany) or MACS Slides with sorted cells were incubated at room temperature (Miltenyi Biotec, Bergisch Gladbach, Germany) technique. (RT) in 3:1 methanol:acetic acid for 15 min and air-dried. The MNCs or selected cell fractions were stained with fluoro- After incubation in hypotonic KCl (0.56%) for 3 min, chrome-conjugated monoclonal antibodies, CD34-FITC 2 × saline sodium citrate (pH 7.0) for 2 min and PBS for 3 min (HPCA-2; Becton Dickinson, Heidelberg, Germany), CD90-PE and pretreatment with pepsin solution, additional washing (in (F15.42; Coulter Immunotech, Hamburg, Germany) or CD90- PBS and PBS-MgCl2), fixation (1% formaldehyde in PBS- PE (5E10; PharMingen, Hamburg, Germany) and isotype con- MgCl2) for 10 min and dehydration procedures (70%, 85% trols (679 1Mc7; Coulter Immunotech). Both CD90 antibodies and 100% ethanol), the hybridization was done according to were found to have almost equal staining performances on the manufacturer’s recommendations. The following DNA the sorting instrument. Staining was done for 30 min at 4°C probes were used: the LSI bcr/abl translocation probe (VYSIS), as described by the manufacturer followed by a washing step the CEP (Chromosome Enumeration DNA FISH Probes) 7 with phosphate-buffered saline (PBS). After setting adjustments spectrum orange probe (locus D7Z1; VYSIS), the CEP 8 spec- with isotype standard controls, CD34+ cells were sorted with Table 1 Amount of CD34 subpopulations in different leukemia samples and normal blood donors No. Diagnosis CD34+/CD90− CD34+/CD90+ CD34 (%) (%) enriched 1 CML ECP 92.4 3.3 yes 2 CML AC 0.6 0.0 no 3 CML AC 19.7 5.0 no 4 CML AC 1.4 0.1 no 5 CML BC 12.0 0.8 no 6 CML BC 9.0 0.0 no 7 CML LCP 0.2 0.0 no 8 CML ECP 4.7 0.05 no 9 CML LCP 4.2 0.0 no 10 CML AC 65.0 0.0 yes 11 CML LCP 20.0 5.0 yes 12 CML BC 60.0 1.5 no 13 CML LCP 59.0 27.0 yes 14 CML LCP 17.1 1.0 yes 15 CML PD 21.8 3.3 yes 16 CML PD 17.1 1.3 yes 17 CML AC 1.0 0.01 no 18 pre B-ALL 43.9 37.5 no 19 T-ALL 0.6 0.0 no 20 sec. AML 72.8 0.12 yes 21 AML relapse 17.9 1.3 no 22 sec.