what can we learn from pre-clinical drug testing in childhood cancer? c. patrick reynolds, md phd...
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What Can We Learn From Pre-Clinical Drug Testing in Childhood Cancer?
C. Patrick Reynolds, MD PhD
ChildrensHospitalLosAngeles
13-cis-Retinoic Acid Causes Sustained Growth Arrest of Neuroblastoma
DAYS
0 14 28 42 56 70 84 98 112126
% C
ON
TR
OL
0
20
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60
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RA RA
0 14 28 42 56 70 84 98 112126
CE
LL
NU
MB
ER
103
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1010
Control
13-cis-RA
13-cis-RA
MYCN Expression
Control
10 M Retinoic Acid
CCG-3891 Event-Free Survival Second Randomization
2/20/02
Principles for In Vitro Testing of Anti-Neoplastic Drugs
• Assay system should have a wide dynamic range, ideally 3 to 4 logs of cell kill, yet allow high throughput
• Cell line panel should employ multiple cell lines, especially those resistant to standard drugs
• Major mechanisms of resistance should be identified and reflected in the cell line panel
• Exposure to drugs should be at clinically achievable levels and schedules
• As hypoxia may antagonize drug action, testing should also be done under hypoxic conditions
Limitations of In Vitro Models For Drug Testing
• Selection of cell cultures for ability to grow in vitro
• Artificially high drug exposure can occur
• Cell culture oxygen conditions far exceed the physiological
• Cell-to-cell contact, especially with normal cells, is not preserved
Cooled CCDCamera
Motorized Stage
Inverted Fluorescence Microscope
DIMSCAN MicroplateCytotoxicity Assay
Viable Cells DetectedWith FDA + Eosin Y
DIMSCAN Cytotoxicity AssayHigh dynamic range (> 4 logs) in 96 Well Plates
DIMSCAN 3.0 Image Thresholding
Original image Thresholded image
Drug Resistance In Human Neuroblastoma Cell Lines
CBDCACDDP
L-PAMDOX
ETOP
FO
LD
RE
SIS
TA
NC
E
DXPD-Ind
PD-BMT
Glutathionine (GSH) is synthesized via glutamylcysteine synthetase (GCS)
GCS is selectively inhibited by BSO, decreasing cellular GSH and enhancing alkylator cytotoxicity
NeuroblastomaButhionine Sulfoximine (BSO) Synergy with L-PAM
Fra
ctio
nal
Su
rviv
al
Melphalan (µM)
Response to Low Dose BSO/L-PAM
November ‘98 December ‘98
Patient # 19: Relapse Post-CCG-3891 Consolidation
Xenograft Models For Drug Testing
• Cell lines responsive and resistant to standard agents should be employed
• Subcutaneous xenografts allow for easy measurement
• Intravenous injection may mimic MRD
• Immunocytochemistry can detect MRD
• New rodent imaging methods may allow for assessment of response in organs, especially lung
Limitations of Rodent Models For Drug Testing
• PK in the mouse can differ from humans
• Adult mice are used for drug testing
• Animal testing is labor-intensive
• Subcutaneous tumors may be quite different than orthotopic tumors
• Transgenic animal models provide “virgin” tumors that have not developed resistance to currently employed drugs
Bone Metastases From Intravenous Injection of the CHLA-255 Neuroblastoma in SCID Mice
Histopathology Micro-CAT High-Resolution
Radiograph
Drug Testing: What Results Should Encourage Pediatric Clinical Trials?
• Multi-log killing of cell lines, including those established at relapse, at clinically achievable drug levels
• Activity against multi-drug resistant cell lines in hypoxia
• Responses in xenografts, ideally in those that are multi-drug resistant
• Significant activity of drug combinations could encourage phase I trials, even if single agents show only modest activity
Drug Testing: What Results Should Discourage Pediatric Clinical Trials?
• Poor activity ( < 1 log cell killing ) at clinically achievable drug levels in multiple cell lines
• Poor activity in xenograft models known to be responsive to standard drugs
• Availability of agents with more promising activity for the same target population
Summary
• Pre-clinical drug testing may be a means for prioritizing new agents
• Validation of existing models should be undertaken retrospectively and prospectively
• Pre-clinical modeling of drug combinations may facilitate design of phase I + II trials