Development and validation of a PCR-based assay for the selection of patients more likely to benefit from therapeutic treatment with alkylating drugs


Dr Vassilis L. Souliotis, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 116 35 Athens, Greece. Tel.: +30 210 727 3733. Fax: +30 210 727 3677. E-mail:



• 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.


• 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/106 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.

AIM 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/106 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%.

CONCLUSION 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.