title: Development and validation of a PCR-based assay for the selection of patients more likely to benefit from therapeutic treatment with alkylating drugs.
abstract: BACKGROUND
  Previous studies have indicated that the levels of DNA damage induced in peripheral blood mononuclear cells by the alkylating drugs melphalan, cisplatin and carboplatin can serve as useful biomarkers predictive of the therapeutic response of cancer patients to these drugs.
  
  
  CONCLUSIONS
  In the present study we developed a quantitative PCR-based assay, for the measurement of DNA damage. The advantages of this methodology are based on: its far greater sensitivity (about 250 times) than the traditional Southern blot-based method (the detection limit is ~10-20 lesions/10(6) nucleotides from the equivalent DNA of ~8000 cells); its simplicity and speed (results obtained within ~8h); its excellent reproducibility, with a coefficient of variance of 10-15% for different DNA preparations from similarly treated cells; its requirement for only minute amounts of material, and; the avoidance of radioisotope labeling. Moreover, emphasis was given to translate basic research findings into clinical practice through the validation of this assay for prediction of clinical outcome in multiple myeloma patients.
  
  
  OBJECTIVE
  In order to develop and validate a simple, sensitive and rapid method for the quantitation of alkylating drug-induced DNA damage.
  
  
  METHODS
  HepG2 cells and blood samples were treated with alkylating drugs (melphalan, cisplatin, carboplatin). Gene-specific damage was examined using Southern blot and a multiplex long quantitative PCR (QPCR) carried out in a 7 kb fragment (part of the p53 gene) and a 0.5 kb fragment (part of the IFN-β1 sequence; internal standard).
  
  
  RESULTS
  The extent of PCR amplification of a p53 fragment was inversely proportional to the treatment concentrations of all anticancer drugs examined, indicating a dose-related inhibition by the DNA adducts formed. Parallel analysis of the same samples using both Southern blot and QPCR showed that the DNA adducts measured by QPCR corresponded to the interstrand cross-links in the case of melphalan, and to total drug-induced lesions in the case of the platinum drugs. The detection limit was ~10-20 lesions/10(6) nucleotides using DNA from ~8000 cells. The method is about 250 times more sensitive than the Southern blot-based method and the reproducibility is excellent, with an intraday coefficient of variance (CV) of 5-9% and an interday CV of 4-12%. Application of the QPCR assay to ex vivo melphalan-treated peripheral blood mononuclear cells from multiple myeloma patients, showed that the positive predictive value of this assay for clinical response to melphalan therapy was 92.9%.
  
  
  CONCLUSIONS
  The PCR-based assay developed in this study can be used for the selection of cancer patients more likely to benefit from therapeutic treatment with alkylating drugs.
authors.affiliation: Institute of Biological Research and Biotechnology, National Hellenic Research Foundation Department of Clinical Therapeutics, Athens University Medical School, Athens, Greece.
authors.forename: Dimitra T
authors.initials: DT
authors.lastname: Stefanou
authors.forename: Hara
authors.initials: H
authors.lastname: Episkopou
authors.forename: Soterios A
authors.initials: SA
authors.lastname: Kyrtopoulos
authors.forename: Aristotelis
authors.initials: A
authors.lastname: Bamias
authors.forename: Maria
authors.initials: M
authors.lastname: Gkotzamanidou
authors.forename: Christina
authors.initials: C
authors.lastname: Bamia
authors.forename: Christina
authors.initials: C
authors.lastname: Liakou
authors.forename: Margarita
authors.initials: M
authors.lastname: Bekyrou
authors.forename: Petros P
authors.initials: PP
authors.lastname: Sfikakis
authors.forename: Meletios-Athanasios
authors.initials: MA
authors.lastname: Dimopoulos
authors.forename: Vassilis L
authors.initials: VL
authors.lastname: Souliotis
mesh_terms: D000328:Adult; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D015139:Blotting, Southern; D016190:Carboplatin / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D018736:DNA Adducts / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects*; D005260:Female; D016158:Genes, p53 / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D016899:Interferon-beta / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D057230:Limit of Detection; D008297:Male; D008558:Melphalan / Q000494:pharmacology; D008875:Middle Aged; D009101:Multiple Myeloma / Q000188:drug therapy / Q000473:pathology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D018579:Patient Selection; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D016896:Treatment Outcome
chemical_list: D018906:Antineoplastic Agents, Alkylating; D018736:DNA Adducts; D016899:Interferon-beta; D016190:Carboplatin; D002945:Cisplatin; D008558:Melphalan