DNA image cytometric measurement as a surrogate end point biomarker in a phase I trial of α-difluoromethylornithine for cervical intraepithelial neoplasia

Cervical intraepithelial neoplasia grade 3 (CIN 3) is considered a high- risk precursor of invasive cervical cancer. α-Difluoromethylornithine (DFMO) is a promising antiproliferative chemopreventive agent. The purpose of this study was to evaluate image cytometric measurement of nuclear DNA (ICM-DNA) as a surrogate end point biomarker (SEB) in a Phase I trial of DFMO for CIN. Thirty patients with CIN 3 were treated with DFMO at five doses, ranging from 0.0625 to 1.0 g/m²/day, for 1 month. Half of the patients had histological responses. Twenty-five pre- and posttreatment cervical biopsy specimens (from 11 responders and 14 nonresponders) were available for this analysis. ICM- DNA was performed on 4-μm sections cut from formalin-fixed tissue blocks and stained with a thionin-SO₂ Feulgen reaction. ICM-DNAs for each case were expressed as normalized measurements (against the nuclear modal absorbance of lymphocytes) of the absorbance of each cell of interest and were presented in bar histograms. The mean normalized summed absorbance (ΣOD(n)) was obtained as a mean histogram of the cell population of interest. Nineteen (76%) of 25 patients had a significant decrease in ΣOD(n) after DFMO treatment. Posttreatment values were significantly lower than pretreatment values in a paired analysis, and responders had significantly lower values than nonresponders. Analyses of different ICM-DNA references, including percentile values of ΣOD(n) distribution, DNA malignancy grade, and 5c exceeding rate, showed a decrease of mean ΣOD(n) during DFMO treatment. In addition, the summed posttreatment ΣOD(n) histograms also showed progressively shorter right shoulders compared with pretreatment histograms in both responders and nonresponders. We concluded that the modulation of ΣOD(n) reflected the chemoprevention effect of DFMO even before morphological changes appeared, and thus, ICM-DNA may be useful as a SEB in chemoprevention trials of DFMO. Additional reasons for using ICM-DNA as a SEB are the relative simplicity of its use, the high accuracy of the results, the low cost of the reagents, the ability to use small tissue samples, and the objectivity and reproducibility of the procedure.