| Abstract |
Purpose: The CEV regimen, carboplatin (C), etoposide (E), and vincristine
(V), is commonly used for treating retinoblastoma (RB). Cyclosporin A
(CsA) has been added as an MDR-1 modulator to potentiate the clinical
activity of CEV. We tested these agents, and also melphalan (M) and
topotecan (T) on a panel of 10 RB cell lines in 20% and 2% O2.
Methods: The DIMSCAN cytotoxicity assay uses digital imaging microscopy
and fluorescein diacetate/eosin Y to quantify viable cells in microplates.
For pre-clinical testing of 2 to 4 anticancer drugs in various
combinations, we have developed a high-throughput (3-4 log dynamic range)
assay using 384-well microplates (DIMSCAN-384). 5/10 cell lines were
established from untreated patients (CHLA-192, 215, 222, 223, 224) and 5
from patients at relapse after chemotherapy (chemo) or radiotherapy (rad)
(CHLA-194, 203, 209, 210, 246). Cell lines with an LC higher than the
clinically achievable plasma concentration (C=3 mug/ml, E=5 mug/ml, V=0.5
mug/ml, M=10 mug/ml, T=0.1 mug/ml) were considered resistant; drug levels
in the eye, and especially in vitreous, are unknown.
Results: Resistance to single agents was seen in 30% (C), 30% (E), 20%
(V), 10% (M), 30% (T) of tested cell lines, with 6 of 10 cell lines
sensitive to all drugs (3 post-chemo, 1 post-rad), and 2 (1 pre-therapy, 1
post-rad) sensitive to only 1 (M or V). Median LC90 values were C=1.2
mug/ml, E=0.8 mug/ml, V=10 ng/ml, M=0.6 mug/ml, T=6.9 ng/ml. Using
DIMSCAN-384, we tested CEV+CsA, and M+T as single agents and in various
combinations in 5 RB cell lines. In 4 of 5 lines M+T did not achieve
synergistic cytotoxicity (P>0.2). CEV was no more active than single
agents in 4 of 5 lines (P>0.15). Single agent CsA was inactive in 5 of 5
tested cell lines (median LC=59 ug/ml). CsA enhanced (p<0.05) the
cytotoxicity of E (in 2 lines) and V (in 3 lines), but not C (in 4/5 lines;
P>0.05). CsA enhanced CEV cytotoxicity (p<0.05) in 3 of 5 cell lines,
achieving an additional 1-2 logs of cell kill over CEV alone. CsA also
enhanced the cytotoxicity of most 2 drug combinations (including those
with C), achieving 0.5-2 logs of additional cell kill over combinations
without CsA, and EV + CsA (in 5/5 lines), CV + CsA (4/5 lines) or CE +
CsA (3/5 lines) were as efective as CEV + CsA (P>0.05). Hypoxia (2% O2)
did not antagonize cytotoxicity of C, E, V, CsA, or any combination of
these agents (P>0.5).
Conclusions: These data suggest that CEV may not be more active than
single agents in many RB, and suggest that at drug levels achievable in
plasma, CsA can significantly enhance the activity of CEV against some RB.
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