title: Mechanisms of topoisomerase I (TOP1) gene copy number increase in a stage III colorectal cancer patient cohort.
abstract: BACKGROUND
  Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH).
  
  
  METHODS
  Nine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a reference probe covering either the centromeric region of chromosome 20 (CEN-20) or chromosome 2 (CEN-2). Tissue sections from 154 chemonaive stage III CRC patients, previously studied with TOP1/CEN-20, were analyzed with TOP1/CEN-2. Relationships between biomarker status and overall survival (OS), time to recurrence (TTR) in CRC and time to local recurrence (LR; rectal cancer only) were determined.
  
  
  RESULTS
  TOP1 aberrations were observed in four cell line metaphases. In all cell lines CEN-2 was found to reflect chromosomal ploidy levels and therefore the TOP1/CEN-2 probe combination was selected to identify TOP1 gene gains (TOP1/CEN-2≥1.5). One hundred and three patients (68.2%) had TOP1 gain, of which 15 patients (14.6%) harbored an amplification (TOP1/CEN-20≥2.0). TOP1 gene gain did not have any association with clinical endpoints, whereas TOP1 amplification showed a non-significant trend towards longer TTR (multivariate HR: 0.50, p = 0.08). Once amplified cases were segregated from other cases of gene gain, non-amplified gene increases (TOP1/CEN-2≥1.5 and TOP1/CEN-20<2.0) showed a trend towards shorter TTR (univariate HR: 1.57, p = 0.07).
  
  
  CONCLUSIONS
  TOP1 gene copy number increase occurs frequently in stage III CRC in a mechanism that often includes CEN-20. Using CEN-2 as a measurement for tumor ploidy levels, we were able to discriminate between different mechanisms of gene gain, which appeared to differ in prognostic impact. TOP1 FISH guidelines have been updated.
authors.affiliation: R&D, Dako A/S, Glostrup, Denmark. dhsmith@dako.com
authors.forename: David Hersi
authors.initials: DH
authors.lastname: Smith
authors.forename: Ib Jarle
authors.initials: IJ
authors.lastname: Christensen
authors.forename: Niels Frank
authors.initials: NF
authors.lastname: Jensen
authors.forename: Bo
authors.initials: B
authors.lastname: Markussen
authors.forename: Maria Unni
authors.initials: MU
authors.lastname: Rømer
authors.forename: Sune Boris
authors.initials: SB
authors.lastname: Nygård
authors.forename: Sven
authors.initials: S
authors.lastname: Müller
authors.forename: Hans Jørgen
authors.initials: HJ
authors.lastname: Nielsen
authors.forename: Nils
authors.initials: N
authors.lastname: Brünner
authors.forename: Kirsten Vang
authors.initials: KV
authors.lastname: Nielsen
mesh_terms: D045744:Cell Line, Tumor; D002889:Chromosomes, Human, Pair 2 / Q000235:genetics; D002890:Chromosomes, Human, Pair 20 / Q000235:genetics; D015331:Cohort Studies; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics* / Q000473:pathology*; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D005260:Female; D018628:Gene Dosage / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D011379:Prognosis
chemical_list: D004264:DNA Topoisomerases, Type I