title: Molecular assessment of erythroid lineage chimerism following nonmyeloablative allogeneic stem cell transplantation.
abstract: OBJECTIVE
  Nonmyeloablative conditioning regimens for allogeneic stem cell transplantation are now commonly used in the treatment of patients with hematologic malignancies. Since this treatment often results in the establishment of mixed hematopoietic chimerism, this approach may also prove to be useful in the treatment of nonmalignant disorders, such as sickle cell disease and thalassemia major. To apply this approach to these diseases, it will be necessary to determine the levels of donor erythropoiesis required to correct hemolysis and ameliorate disease symptoms. Current methods for measuring hematopoietic chimerism are based on DNA polymorphisms that distinguish recipient from donor. These methods accurately measure donor leukocyte engraftment but do not quantify the relative contributions of recipient and donor erythropoiesis following transplant.
  
  
  METHODS
  To specifically measure erythroid-lineage chimerism, we used pyrosequencing of the sickle cell mutation to quantify the relative levels of normal and sickle beta-globin mRNA in patient samples. Results of beta-globin RNA chimerism were compared to assessment of beta-globin DNA chimerism as well as analysis of short tandem repeat (STR) polymorphisms, cytogenetics, and hemoglobin electrophoresis.
  
  
  RESULTS
  Donor engraftment was measured in two adult patients following nonmyeloablative stem cell transplant for sickle cell disease. In Patient 1, 25 to 30% of peripheral leukocytes were donor derived after day 41. In contrast, more than 55% of peripheral blood beta-globin mRNA was of donor origin, and these results correlated with posttransplant clinical improvement. Patient 2 achieved 40 to 50% donor leukocyte engraftment from day 33 onward. This was associated with 70 to 100% peripheral blood donor beta-globin mRNA.
  
  
  CONCLUSIONS
  These studies demonstrate that relatively low levels of donor leukocyte engraftment can be associated with higher levels of donor erythropoiesis and with significant clinical improvement. Pyrosequencing of lineage-specific mRNA directly measures functional reconstitution of donor cells and provides valuable information that can affect clinical decisions in patients with nonmalignant diseases following allogeneic transplant.
authors.affiliation: Departments of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. cwu@partners.org
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Wu
authors.forename: Ephraim P
authors.initials: EP
authors.lastname: Hochberg
authors.forename: Shelby A
authors.initials: SA
authors.lastname: Rogers
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: Kutok
authors.forename: Melinda
authors.initials: M
authors.lastname: Biernacki
authors.forename: Alessandra F
authors.initials: AF
authors.lastname: Nascimento
authors.forename: Peter
authors.initials: P
authors.lastname: Marks
authors.forename: Kenneth
authors.initials: K
authors.lastname: Bridges
authors.forename: Jerome
authors.initials: J
authors.lastname: Ritz
mesh_terms: D000328:Adult; D019070:Cell Lineage; D004920:Erythropoiesis; D005260:Female; D005914:Globins / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000032:analysis; D018183:Transplantation Chimera; D014184:Transplantation, Homologous
chemical_list: D012333:RNA, Messenger; D005914:Globins