title: Development and validation of an immunoassay for quantification of topoisomerase I in solid tumor tissues.
abstract: BACKGROUND
  Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors.
  
  
  RESULTS
  We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998.
  
  
  CONCLUSIONS
  Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Support Directorate, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America. pfistert@mail.nih.gov
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pfister
authors.forename: Melinda
authors.initials: M
authors.lastname: Hollingshead
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kinders
authors.forename: Yiping
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yvonne A
authors.initials: YA
authors.lastname: Evrard
authors.forename: Jiuping
authors.initials: J
authors.lastname: Ji
authors.forename: Sonny A
authors.initials: SA
authors.lastname: Khin
authors.forename: Suzanne
authors.initials: S
authors.lastname: Borgel
authors.forename: Howard
authors.initials: H
authors.lastname: Stotler
authors.forename: John
authors.initials: J
authors.lastname: Carter
authors.forename: Raymond
authors.initials: R
authors.lastname: Divelbiss
authors.forename: Shivaani
authors.initials: S
authors.lastname: Kummar
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Parchment
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Tomaszewski
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
mesh_terms: D000818:Animals; D001706:Biopsy; D045744:Cell Line, Tumor; D002986:Clinical Trials as Topic; D004264:DNA Topoisomerases, Type I / Q000097:blood / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005260:Female; D007118:Immunoassay / Q000379:methods*; D007546:Isoquinolines / Q000737:chemistry / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013997:Time Factors; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology; D019772:Topotecan / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: D007546:Isoquinolines; D059004:Topoisomerase I Inhibitors; D019772:Topotecan; D011065:Poly(ADP-ribose) Polymerases; D004264:DNA Topoisomerases, Type I; C039109:isoquinoline