title: BCR/ABL-negative primitive progenitors suitable for transplantation can be selected from the marrow of most early-chronic phase but not accelerated-phase chronic myelogenous leukemia patients.
abstract: We have previously reported that selection of marrow cells on the basis of the CD34+HLA-DR- phenotype (34+DR-) may result in the recovery of Philadelphia chromosome (Ph)- and BCR/ABL-negative long-term culture-initiating cells (LTC-IC) in selected patients with chronic myelogenous leukemia (CML). We now present data on 27 early chronic-phase ([ECP] studied within 1 year after diagnosis) and 23 advanced-phase ([AP] late chronic phase, ie, studied >1 year from diagnosis, or accelerated phase) CML patients. Fluorescence-activated call-sorting (FACS)-selected 34+DR- and 34+DR+ cells were subjected to reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization. These cells were also cultured in long-term bone marrow culture for 1 to 5 weeks to examine the number of LTC-IC and the presence or absence of the BCR/ABL gene rearrangement in progeny of primitive LTC-IC. The number of 34+DR- cells and LTC-IC present in ECP CML marrow was similar to that in normal (NL) marrow, whereas the numbers were reduced in AP CML. Furthermore, 34+DR- cells from more than 80% of ECP CML patients were BCR/ABL mRNA- and Ph-negative and contained only BCR/ABL mRNA- and Ph-negative LTC-IC, whereas 34+DR- cells and LTC-IC from less than 40% of AP CML patients were BCR/ABL mRNA- and Ph-negative. In contrast to NL marrow, 34+DR+ cells from CML marrow, irrespective of clinical stage, contained large numbers of LTC-IC. CML 34+DR+ cells and LTC-IC were BCR/ABL mRNA- and Ph-positive. Since these studies suggested that a population of primitive progenitors that are Ph-negative can be selected from steady-state marrow in some ECP CML patients, we determined if similar results could be obtained when large quantities of marrow sufficient for transplantation are processed. We demonstrate that 1 to 3 x 10(5) BCR/ABL mRNA-negative 34+DR- cells/kg recipient body weight, containing only BCR/ABL mRNA-negative LTC-IC, can be obtained from a 2- to 2.5-L marrow collection by sequential COBE Spectra apheresis (COBE BCT, Lakewood, CO), CD34+ enrichment using the CEPRATE SC Cell-Concentrator (CellPro, Bothell, WA), and high-speed FACS. Thus, large-scale selection of a BCR/ABL mRNA- and Ph-negative 34+DR- cell population is possible in a fraction of chronic-phase CML patients, in whom these cells could be used to reconstitute the hematopoietic compartment following autologous transplantation.
authors.affiliation: Division of Hematology and the Department of Laboratory Medicine, University of Minnesota, Minneapolis 55455, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Verfaillie
authors.forename: R
authors.initials: R
authors.lastname: Bhatia
authors.forename: W
authors.initials: W
authors.lastname: Miller
authors.forename: F
authors.initials: F
authors.lastname: Mortari
authors.forename: V
authors.initials: V
authors.lastname: Roy
authors.forename: S
authors.initials: S
authors.lastname: Burger
authors.forename: J
authors.initials: J
authors.lastname: McCullough
authors.forename: K
authors.initials: K
authors.lastname: Stieglbauer
authors.forename: G
authors.initials: G
authors.lastname: Dewald
authors.forename: S
authors.initials: S
authors.lastname: Heimfeld
authors.forename: J S
authors.initials: JS
authors.lastname: Miller
authors.forename: P B
authors.initials: PB
authors.lastname: McGlave
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D001781:Blood Component Removal / Q000379:methods*; D001853:Bone Marrow / Q000473:pathology*; D002452:Cell Count; D002478:Cells, Cultured / Q000637:transplantation; D016044:Fusion Proteins, bcr-abl / Q000032:analysis*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015465:Leukemia, Myeloid, Accelerated Phase / Q000473:pathology* / Q000628:therapy; D015466:Leukemia, Myeloid, Chronic-Phase / Q000473:pathology* / Q000628:therapy; D009363:Neoplasm Proteins / Q000032:analysis*; D014411:Neoplastic Stem Cells / Q000737:chemistry*; D010677:Philadelphia Chromosome; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis
chemical_list: D018952:Antigens, CD34; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016044:Fusion Proteins, bcr-abl