1. What Can We Learn From Pre-Clinical Drug Testing in Childhood Cancer?
C. Patrick Reynolds, MD PhD
ChildrensHospitalLosAngeles

2. 13-cis-Retinoic Acid Causes Sustained Growth Arrest of Neuroblastoma
Control
10 mM Retinoic Acid

3. CCG-3891 Event-Free Survival Second Randomization
2/20/02

4. 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

5. 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

6. High dynamic range (> 4 logs) in 96 Well Plates
DIMSCAN Cytotoxicity Assay
High dynamic range (> 4 logs) in 96 Well Plates
DIMSCAN Microplate
Cytotoxicity Assay
Viable Cells Detected
With FDA + Eosin Y
Cooled
CCD
Camera
Motorized Stage
Inverted Fluorescence Microscope

7. DIMSCAN 3.0 Image Thresholding
Original image
Thresholded image

8. Drug Resistance In Human Neuroblastoma Cell LinesDX
PD-Ind
PD-BMT

9. Buthionine Sulfoximine (BSO) Synergy with L-PAM
Neuroblastoma
Buthionine Sulfoximine (BSO) Synergy with L-PAM
Glutathionine (GSH) is synthesized via glutamylcysteine synthetase (GCS)
GCS is selectively inhibited by BSO, decreasing cellular GSH and enhancing alkylator cytotoxicity
Fractional Survival
Melphalan (µM)

10. Response to Low Dose BSO/L-PAM
Patient # 19: Relapse Post-CCG-3891 Consolidation
November ‘ December ‘98

11. 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

12. 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

13. Bone Metastases From Intravenous Injection of the CHLA-255 Neuroblastoma in SCID Mice
High-Resolution
Radiograph
Histopathology
Micro-CAT

14. 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

15. 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

16. 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