What Can We Learn From Pre-Clinical Drug Testing in Childhood Cancer?

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What Can We Learn From Pre-Clinical Drug Testing
in Childhood Cancer?

• C. Patrick Reynolds, MD PhD

ChildrensHospitalLosAngeles
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13-cis-Retinoic Acid Causes Sustained Growth
Arrest of Neuroblastoma
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CCG-3891 Event-Free Survival Second Randomization
2/20/02
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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

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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

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DIMSCAN Cytotoxicity Assay
High dynamic range (gt 4 logs) in 96 Well Plates
DIMSCAN Microplate Cytotoxicity Assay
Viable Cells Detected With FDA Eosin Y
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DIMSCAN 3.0 Image Thresholding
Original image
Thresholded image
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Drug Resistance In Human Neuroblastoma Cell Lines
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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)
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Response to Low Dose BSO/L-PAM
Patient 19 Relapse Post-CCG-3891 Consolidation
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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

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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

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

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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

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Drug Testing What Results Should Discourage
Pediatric Clinical Trials?

• Poor activity ( lt 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

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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