title: Pharmacological purging of minimal residual disease from peripheral blood stem cell collections of acute myeloblastic leukemia patients: preclinical studies.
abstract: In this paper we describe an experimental model for ex vivo purging of contaminating tumor cells from peripheral blood stem cell (PBSC) collections obtained from patients with acute myeloblastic leukemia (AML). We studied the combination of the alkylating agent nitrogen mustard (NM; concentrations ranging from 0.25 to 1.25 microg/mL) and etoposide (VP-16; constant dose of 20 microg/mL), and the conventional cyclophosphamide (Cy)-derivative mafosfamide (concentrations: 20-175 microg/mL).
  
  
  OBJECTIVE
  1) To compare the toxicity of the purging protocols on bone marrow (BM) and circulating trilineage precursors collected from normal donors after priming with granulocyte colony-stimulating factor (G-CSF) or after complete remission (CR) consolidation chemotherapy and G-CSF (leukemic patients); 2) to demonstrate the survival of very primitive hematopoietic progenitors (LTC-IC) in the peripheral blood (PB) and the BM after pharmacological treatment; and 3) to evaluate the antineoplastic efficacy of purging protocols on PBSC collections using 3 well-established leukemic cell lines. Our results demonstrated that the toxicity on BM and PB progenitor cells could be correlated with the complete killing of committed granulocyte-macrophage colony-forming units (CFU-GMs) and erythroid precursors (BFU-Es), a condition reached at the concentration of 1.5 microg/mL of NM (in addition to 20 microg/mL of VP-16) and 175 microg/mL of mafosfamide. Notably, early and late megakaryocyte progenitor cells (CFU-MKs and BFU-MKs, respectively) showed higher sensitivity to NM/VP-16, but not to mafosfamide, than did CFU-GMs and BFU-Es. The dose of NM capable of inhibiting 95% of CFU-MKs and BFU-MKs (ID95) was 0.75 microg/mL. After incubation with the same dose of NM, the recovery of CFU-GMs and BFU-Es was 20 +/- 8% SD and 25 +/- 10% SD, respectively (p < 0.05). Long-term liquid cultures showed the recovery of primitive hematopoietic cells after incubation with the highest concentrations of NM/VP-16 and mafosfamide, with no significant differences between PB and BM samples. Under the same experimental conditions, we observed a more than 5-log reduction of contaminating leukemic cell lines (i.e., K-562, KG-1, and HL-60). In conclusion, we demonstrated that NM/VP-16 and mafosfamide purging agents are capable of killing leukemic cell lines that contaminate leukapheresis products from patients with AML, whereas an acceptable proportion of primitive LTC-IC is spared. Moreover, despite the different kinetic and functional profile of mobilized and steady-state BM progenitors, we did not observe any difference in toxicity of antineoplastic agents on hematopoietic cells at different levels of differentiation. These data suggest that pharmacological strategies developed for eliminating minimal residual disease (MRD) from BM autografts can be effectively and safely applied to circulating stem cell harvests.
authors.affiliation: Institute of Hematology and Oncology, University of Bologna, Italy.
authors.forename: M R
authors.initials: MR
authors.lastname: Motta
authors.forename: S
authors.initials: S
authors.lastname: Mangianti
authors.forename: S
authors.initials: S
authors.lastname: Rizzi
authors.forename: M
authors.initials: M
authors.lastname: Ratta
authors.forename: E
authors.initials: E
authors.lastname: Campanini
authors.forename: A
authors.initials: A
authors.lastname: Fortuna
authors.forename: M
authors.initials: M
authors.lastname: Fogli
authors.forename: S
authors.initials: S
authors.lastname: Tura
authors.forename: R M
authors.initials: RM
authors.lastname: Lemoli
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D001854:Bone Marrow Cells / Q000187:drug effects; D003520:Cyclophosphamide / Q000008:administration & dosage / Q000031:analogs & derivatives*; D005047:Etoposide / Q000008:administration & dosage*; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000097:blood / Q000188:drug therapy* / Q000473:pathology; D008466:Mechlorethamine / Q000008:administration & dosage*; D018365:Neoplasm, Residual / Q000188:drug therapy*; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D008466:Mechlorethamine; C048341:mafosfamide; D005047:Etoposide; D003520:Cyclophosphamide