Article Figures & Data
Figures
- Fig. 1.
Induction of γH2AX in topotecan-responsive and nonresponsive xenograft tumor biopsies and hair follicles from mice treated with topotecan. A, assay specificity for γH2AX immunostaining of untreated mouse small intestine (negative control), untreated mouse testis (positive control), and mouse small intestine 2 hours after treatment with 0.32 MTD topotecan. Bottom right, a representative section of γH2AX staining in hair follicles isolated from skin snip samples taken 2 hours after treatment with 0.32 MTD topotecan. Magnification, ×400. B, γH2AX-stained topotecan-responsive A375 xenograft biopsy samples 2 hours post-treatment with vehicle, or 0.03, 0.1, or 0.32 MTD topotecan. Magnification, ×400. C, γH2AX staining in nonresponsive SK-MEL-28 xenograft biopsy (top) and hair follicle (bottom) samples from the same animal 5 hours post-treatment with 1 MTD topotecan. Magnification, ×200. D, mean %NAP for γH2AX in A375 xenograft biopsies from mice treated with vehicle control, or 0.1 or 0.32 MTD topotecan collected 1, 2, 4, and 7 hours post-dose; four mice per cohort. The mean %NAP for all topotecan-treated groups was statistically different from vehicle, with a significance level of P ≤ 0.001, as determined by Student's t-test. cy, small intestine crypts; sg, spermatagonia; hf, hair follicle; sm, smooth muscle.
- Fig. 2.
Interlaboratory method validation. Comparison of 75 duplicate slides processed for γH2AX immunofluorescence at two independent laboratories, the National Clinical Target Validation Laboratory (NCTVL) and the Pathology/Histotechnology Laboratory (PHL). Image quantitation and analysis were run by both laboratories independently, and %NAP for γH2AX values were compared across sites.
- Fig. 3.
Tumor growth delay is observed after treatment with increasing doses of NSC 724988 or topotecan. Mice with A375 tumor xenografts were treated with topotecan (1.5 or 4.7 mg/kg) or NSC 724998 (4, 8, or 12 mg/kg) for one cycle given once daily for 5 days or with NSC 724998 (16 mg/kg) for two cycles given once daily for 5 days (17-day rest between cycles). A, xenograft tumor volume was measured throughout the study period. Data points, median ± SE; 8 mice per treatment group and 16 mice per control group. B, regression analysis of γH2AX %NAP measurements 4 hours post-dose on day 5 of cycle 1 compared with tumor volumes on day 23 of cycle 1. Dashed vertical line, average tumor volume at staging was 200 mm3. Data points, median ± SE; 4 mice per treatment group and 8 mice per control group.
- Fig. 4.
A significant γH2AX response was observed compared with vehicle control at all doses of NSC 724998 in A375 xenograft tumor biopsies collected 4 and 7 hours post-treatment. Box plots, interquartile range and median %NAP for γH2AX for three to five mice per dose group per time point. Biopsies were collected 1, 2, 4, and 7 hours after a single dose of 0.16 to 0.67 MTD NSC 724998 or vehicle control. *, treatment cohort mean was statistically different from vehicle mean at same time point, with a significance level of P < 0.05, as determined by Student's t-test.
Tables
- Table 1.
Antitumor activity of NSC 724998 and topotecan in A375 xenografts
No. of mice* Drug-related deaths† Maximum % mean body weight loss (d) Tumor growth inhibition Optimal % T/C (d) Growth delay % (T-C)/C Net log cell kill Vehicle 16 0 0.9 (12) NSC 724998 16 mg/kg QD×5‡ 8 0 7.2 (12) 1 (19) 259 0.8 12 mg/kg QD×5 8 0 7.1 (12) 3 (19) 116 1.4 8 mg/kg QD×5 8 0 3.6 (12) 12 (15) 68 0.6 4 mg/kg QD×5 8 0 3.2 (12) 36 (15) 28 −0.1 Topotecan 4.7 mg/kg QD×5 8 2 28.4 (15) 3 (15) 143 1.8 1.5 mg/kg QD×5 8 0 6.4 (12) 13 (15) 54 0.3 Abbreviations: QD×5, treated for 5 sequential days at designated dose; T, treated group; C, control group; d, day.
↵*No mice were tumor-free by study day 70.
↵†A death is considered treatment-related if the animal dies within 15 days of the last treatment and either the tumor weight is less than the lethal burden in the control mice or its net body weight loss at death is 20% greater than the mean net weight change in the control animals at death or sacrifice.
↵‡The NSC 724998 16-mg/kg treatment group was treated daily for 5 days for two cycles with a 17-day rest between treatments; all other groups were treated for one cycle.
- Table 2.
Intergroup statistical differences in γH2AX response in A375 xenograft biopsies collected 2 hours post-dose
NSC 724998* Vehicle 0.05 MTD 0.1 MTD 0.33 MTD 1 MTD Vehicle — — — — — 0.05 MTD 0.026 — — — — 0.1 MTD 0.017 NS — — — 0.33 MTD 0.001 NS NS — — 1 MTD 0.034 0.0381 NS 0.090 — NSC 725776* Vehicle 0.05 MTD 0.1 MTD 0.33 MTD 1 MTD Vehicle — — — — — 0.05 MTD 0.010 — — — — 0.1 MTD 0.103 NS — — — 0.33 MTD 0.002 NS NS — — 1 MTD 0.034 NS 0.038 NS — NOTE: One-way ANOVA, with the significance level (α) set at 0.05, two-sided, was used to determine P-values for statistical differences across treatment groups. P-values were calculated with the use of pairwise comparisons of γH2AX response to identify specific intergroup response differences. P-values ≤ 0.10 are reported due to the small group sizes. P-values > 0.10 are reported as NS.
Abbreviation: NS, not significant.
↵*Four mice per group.
Supplementary Data
Supplementary Figures S1-S6; Supplementary Data; Supplementary Tables S1-S3.
Files in this Data Supplement:
- Supplementary Data - Supplementary Figures S1-S6; Supplementary Data; Supplementary Tables S1-S3.
Volume 16, Issue 22
