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 Control 10 mM 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
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
DIMSCAN 3.0 Image Thresholding Original image Thresholded image
Drug Resistance In Human Neuroblastoma Cell Lines DX PD-Ind PD-BMT
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)
Response to Low Dose BSO/L-PAM Patient # 19: Relapse Post-CCG-3891 Consolidation November ‘98 December ‘98
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 High-Resolution Radiograph Histopathology Micro-CAT
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