19 It has the advantage of requiring few cells, and, because analysis can be made at a single-cell level, the method allows the detection of heterogeneity of response within a cell population. The single-cell gel electrophoresis (Comet) assay was originally developed to measure DNA strand breaks in a single cell population 18and has been modified to allow the study of ICL formation and repair. 17 Alkaline elution, however, requires a large number of cells and is not easily adapted for in vivo studies. This mechanism may have implications for MM patients undergoing melphalan therapy.ĭNA ICL formation can be measured in plasmid DNA by using an agarose gel–based method, 16 and the formation and repair of ICL in intact cells can be followed by using alkaline filter elution.
These findings suggest that ICL repair may be an important mechanism by which melphalan resistance emerges after autologous SCT or oral therapy. In vitro sensitivity to melphalan in plasma cells was found to correlate with ICL repair. However, marked differences in ICL repair were observed: cells from naive patients showed no repair, whereas those from treated patients exhibited between 42% and 100% repair at 40 hours. Similar levels of dose-dependent DNA interstand crosslinking were observed in cells from both melphalan-naive and -treated patients. By using a modification of the single-cell gel electrophoresis (Comet) assay, we have measured formation and repair of DNA ICL in plasma cells from melphalan- naive and melphalan-treated patients (ie, those who have relapsed after a melphalan-conditioned autologous SCT or oral melphalan therapy). Melphalan produces a number of DNA adducts with the DNA interstrand crosslink (ICL) considered to be the critical cytotoxic lesion.
Although disease relapse is the major cause of death after a melphalan-conditioned autograft, the mechanism remains unclear. Melphalan is widely used as a preparative agent in patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (SCT).