title: A novel phenotypic method to determine fludarabine triphosphate accumulation in T-lymphocytes from hematopoietic cell transplantation patients.
abstract: OBJECTIVE
  Fludarabine is an integral anticancer agent for patients with chronic lymphocytic leukemia (CLL) and those receiving conditioning regimens prior to allogeneic hematopoietic cell transplantation (HCT). An individual's response to fludarabine may be influenced by the amount of CD4(+) and CD8(+) T-lymphocyte suppression. Fludarabine undergoes cellular uptake and activation to form the cytotoxic metabolite, fludarabine triphosphate (F-ara-ATP).
  
  
  METHODS
  We have previously developed a highly sensitive LC-MS method to quantitate intracellular F-ara-ATP concentrations in a leukemic cell line. However, quantitation of F-ara-ATP concentrations within CD4(+) and CD8(+) T-lymphocytes from pharmacokinetic blood samples obtained from patients receiving fludarabine therapy is not feasible because of the limited number of T-lymphocytes that can be isolated from each blood sample. Thus, we sought to determine F-ara-ATP accumulation after ex vivo exposure of freshly isolated human CD4(+) or CD8(+) T-lymphocytes to fludarabine. The method was optimized in T-lymphocytes obtained from healthy volunteers, and proved to be a feasible method to determine F-ara-ATP accumulation in patients undergoing HCT.
  
  
  RESULTS
  Considerable variability was observed in F-ara-ATP accumulation in HCT patients (10.5- and 12.5-fold in CD4(+) and CD8(+) cells, respectively), compared to healthy volunteers (1.6- and 1.9-fold in CD4(+) and CD8(+) cells, respectively). Larger variability was also observed in gene expression of transporters and enzymes involved in F-ara-ATP accumulation in HCT patients; however, F-ara-ATP accumulation was not correlated with gene expression, which is in agreement with previous studies.
  
  
  CONCLUSIONS
  The quantitation of F-ara-ATP accumulation in T-lymphocytes provides a novel tool to evaluate patient sensitivity to fludarabine. This tool can be used in future studies to evaluate whether intracellular F-ara-ATP accumulation is associated with efficacy and/or toxicity in patients receiving fludarabine.
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors.forename: Erica L
authors.initials: EL
authors.lastname: Woodahl
authors.forename: Joanne
authors.initials: J
authors.lastname: Wang
authors.forename: Shelly
authors.initials: S
authors.lastname: Heimfeld
authors.forename: Brenda M
authors.initials: BM
authors.lastname: Sandmaier
authors.forename: Paul V
authors.initials: PV
authors.lastname: O'Donnell
authors.forename: Brian
authors.initials: B
authors.lastname: Phillips
authors.forename: Linda
authors.initials: L
authors.lastname: Risler
authors.forename: David K
authors.initials: DK
authors.lastname: Blough
authors.forename: Jeannine S
authors.initials: JS
authors.lastname: McCune
mesh_terms: D000328:Adult; D000368:Aged; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000627:therapeutic use; D002853:Chromatography, Liquid; D005260:Female; D015870:Gene Expression; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D010641:Phenotype; D013601:T-Lymphocytes / Q000378:metabolism*; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000627:therapeutic use
chemical_list: D000970:Antineoplastic Agents; D014740:Vidarabine; C024352:fludarabine