Clinical Cancer Research, Vol 4, Issue 3 545-557, Copyright © 1998 by American Association for Cancer Research

ARTICLES

Pharmacodynamics of topoisomerase I inhibition: Western blot determination of topoisomerase I and cleavable complex in patients with upper gastrointestinal malignancies treated with topotecan

L Liebes, M Potmesil, T Kim, D Pease, M Buckley, D Fry, J Cho, H Adler, K Dar, A Zeleniuch-Jacquotte and H Hochster
Kaplan Cancer Center, New York University Medical Center, New York 10016, USA.

Analogues of camptothecins are specific inhibitors of eukaryotic DNA topoisomerase I (topo I) that lead to DNA damage and, eventually, cellular cytotoxicity. Camptothecin analogues bind to this target enzyme in the course of its normal function and stabilize the DNA-enzyme adduct to form a "cleavable complex." Preclinical experiments using Western blot analyses have shown cleavable complex formation to be the key intermediate step in topo I inhibition. In this series of experiments, it was our goal to convert this laboratory technique into a useful clinical assay, allowing measurement of the target enzyme and detection of the key intermediate in clinical specimens taken from patients being treated with the topo I inhibitor topotecan. Because available antibodies were not sufficiently sensitive at the start of this project, we identified a highly specific human SCL-70 antibody from a patient with scleroderma, which allowed quantitative determination of topo I copy number in HeLa and HT-29 cell lines. Additional refinements of the Western blot technique were accomplished to improve signal:noise ratio. In surgical tumor specimens, we found the median topo I level to be 30.1 x 10(5) copies/cell for gastric adenocarcinomas, compared to 18.4 x 10(5) copies/cell for normal gastric mucosae in the same samples. For lung adenocarcinoma, the median protein level was 21.5 x 10(5) copies/cell, compared with the normal tissue counterpart protein level of 12.7 x 10(5) copies/cell. The median tumor:normal ratios from paired samples of these tumor types were 1.51 and 1.84, respectively. As part of a Phase II study evaluating the efficacy of topotecan (1.5-2.0 mg/m2 daily for 5 days) in upper gastrointestinal malignancies, we obtained tumor and normal mucosa biopsies in 11 patients with gastric or esophageal cancer, 30 min after administration on day 4 or 5. Three patients with gastric adenocarcinoma had stable disease as their best response, with the remainder of patients progressing. Improvement in Western blotting methodology allowed the quantitation of topo I levels in these gastric and esophageal cancer biopsies, which could be augmented by brief heating to release complexed topo I. We were also able to directly visualize high molecular weight topo I-containing bands, which were shown to be cleavable complexes by heat reversal, with restoration of the topo I Mr 100,000 band. Using this heat reversal technique, we determined the presence of cleavable complex in a total of 7 of 11 patient biopsy samples (5 tumors and 2 normal mucosae). In patients treated with topotecan on this dose and schedule, we determined that a median of 73% of the total tumor topo I was involved in cleavable complex (range, 18.3-91%). The intensity of the Mr 100,000 topo I band in biopsy specimens of patients receiving topotecan represented "free" or noncomplexed topo I. The median copy number for the residual, noncomplexed topo I (n = 11) was 7.36 x 10(5) copies/cell, significantly less than the median of 30.1 x 10(5) copies/cell for random tumor specimens from patients with gastric adenocarcinomas (P < 0.001). Pharmacodynamic analysis demonstrated a negative correlation between the noncomplexed topo I copy number and topotecan area under the curve (Spearman rank test: r(s) = -0.81, P = 0.003). Nonlinear regression analyses of these data were best fit with an inhibitory maximum effect model, yielding parameter estimates for Emax and EC50 of 29.3 x 10(5) copies/cell (coefficient of variation = 22%) and 43.1 ng x h/ml (coefficient of variation = 27%), respectively. Through a series of careful modifications and refinements, we have improved the Western blot assay for topo I for use in clinical monitoring. We have demonstrated the ability to directly visualize cleavable complex in patients being treated with topo I inhibitor therapy and have directly quantitated free topo I, as well as the key topo I intermediate (cleavable complex), in biopsy specimens obtained from pat

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