Abstract
Inter-individual differences in DNA adduct formation and repair influence the response to melphalan treatment, however, further clinical investigation of this variability requires a logistically feasible and reproducible bioassay. Our improved fluorescence-based QPCR-block assay is robust, has good precision, and improved throughput. It also incorporates direct PCR amplification from melphalan exposed PBMC using commercially available blood tubes and extraction kits to maximise the utility of this assay for future clinical studies. Using this assay we have demonstrated reproducible inter-individual differences in melphalan-induced QPCR-block across individual PBMC donors. As proof-of-principle we assessed nine healthy donors and found a 7.8 fold range in sensitivity following exposure of PBMC ex vivo. This likely reflects differences in melphalan transport into cells as well as differences in DNA adduct repair proficiency. This improved bioassay may be useful for assessment of these processes in patients about to receive melphalan treatment.
Highlights
Inter-individual differences in DNA adduct formation and repair influence the response to melphalan treatment, further clinical investigation of this variability requires a logistically feasible and reproducible bioassay
We report comparison of the published method with an improved, higher throughput assay and demonstrate the ability of this assay to detect inter-individual differences in DNA adduct formation ex vivo in PBMC from healthy donors
Initial experiments were undertaken on purified, untreated genomic DNA pooled from six volunteers
Summary
Inter-individual differences in DNA adduct formation and repair influence the response to melphalan treatment, further clinical investigation of this variability requires a logistically feasible and reproducible bioassay. Our improved fluorescence-based QPCR-block assay is robust, has good precision, and improved throughput It incorporates direct PCR amplification from melphalan exposed PBMC using commercially available blood tubes and extraction kits to maximise the utility of this assay for future clinical studies. Using this assay we have demonstrated reproducible interindividual differences in melphalan-induced QPCR-block across individual PBMC donors. As proof-ofprinciple we assessed nine healthy donors and found a 7.8 fold range in sensitivity following exposure of PBMC ex vivo This likely reflects differences in melphalan transport into cells as well as differences in DNA adduct repair proficiency. A functional assay of adduct formation could provide a useful biomarker to both assess DNA adduct formation and repair proficiency, and to predict response to melphalan treatment
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