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

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

40

60

80

100

RA RA

0 14 28 42 56 70 84 98 112126

CE

LL

NU

MB

ER

103

104

105

106

107

108

109

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

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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