title: Monitoring drug-induced gammaH2AX as a pharmacodynamic biomarker in individual circulating tumor cells.
abstract: OBJECTIVE
  Circulating tumor cells (CTC) in peripheral blood of patients potentially represent a fraction of solid tumor cells available for more frequent pharmacodynamic assessment of drug action than is possible using tumor biopsy. However, currently available CTC assays are limited to cell membrane antigens. Here, we describe an assay that directly examines changes in levels of the nuclear DNA damage marker gammaH2AX in individual CTCs of patients treated with chemotherapeutic agents.
  
  
  METHODS
  An Alexa Fluor 488-conjugated monoclonal gammaH2AX antibody and epithelial cancer cell lines treated with topotecan and spiked into whole blood were used to measure DNA damage-dependent nuclear gammaH2AX signals in individual CTCs. Time-course changes in both CTC number and gammaH2AX levels in CTCs were also evaluated in blood samples from patients undergoing treatment.
  
  
  RESULTS
  The percentage of gammaH2AX-positive CTCs increased in a concentration-dependent manner in cells treated with therapeutically relevant concentrations of topotecan ex vivo. In samples from five patients, percent gammaH2AX-positive cells increased post-treatment from a mean of 2% at baseline (range, 0-6%) to a mean of 38% (range, 22-64%) after a single day of drug administration; this increase was irrespective of increases or decreases in the total CTC count.
  
  
  CONCLUSIONS
  These data show promise for monitoring dynamic changes in nuclear biomarkers in CTCs (in addition to CTC count) for rapidly assessing drug activity in clinical trials of molecularly targeted anticancer therapeutics as well as for translational research.
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Science Applications International Corporation, Frederick, MD, USA.
authors.forename: Lihua H
authors.initials: LH
authors.lastname: Wang
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pfister
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Parchment
authors.forename: Shivaani
authors.initials: S
authors.lastname: Kummar
authors.forename: Larry
authors.initials: L
authors.lastname: Rubinstein
authors.forename: Yvonne A
authors.initials: YA
authors.lastname: Evrard
authors.forename: Martin E
authors.initials: ME
authors.lastname: Gutierrez
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Murgo
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Tomaszewski
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kinders
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D014408:Biomarkers, Tumor / Q000097:blood*; D045744:Cell Line, Tumor; D004249:DNA Damage; D016903:Drug Monitoring / Q000379:methods*; D006657:Histones / Q000097:blood*; D006801:Humans; D009369:Neoplasms / Q000097:blood* / Q000188:drug therapy; D009360:Neoplastic Cells, Circulating / Q000737:chemistry*; D015588:Observer Variation
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; C468783:H2AX protein, human; D006657:Histones