development of assays for use in patient response measurement in phase 0 trials
DESCRIPTION
Development of Assays for Use in Patient Response Measurement in Phase 0 Trials. Robert Kinders Ralph Parchment Pharmacodynamic Assay Development and Implementation Section, and Laboratory of Human Toxicology & Pharmacology SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702 - PowerPoint PPT PresentationTRANSCRIPT
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Development of Assays for Use in Patient Response Measurement in
Phase 0 Trials
Robert KindersRalph Parchment
Pharmacodynamic Assay Development and Implementation Section, and Laboratory of Human Toxicology & Pharmacology
SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702
Funded by Contract N01-CO-12400
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Assay Design Parameters for Phase 0
• Quantitative assay readouts are essential to meet protocol endpoints– Accuracy by recovery, interfering substances, cross-reactivity
and dilution linearity– LLQ, slope and dynamic range must be consistent with expected
signal level in an 18-gauge needle biopsy or 2.5-10 E5 PBMC– Specimen turnaround in 24 hours
• Assay must yield consistent results over at least a year of clinical testing
• Controls and calibrators are critical – Calibrators should be available, stable, quantified, characterized– Controls should mimic specimen and be run in every assay
• PAR assay uses cell extracts • Specific antibody-stained biopsies used in γ-H2AX assay
• Reagent sources and an alternate vendor should be identified
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Tumor Extract Dilution Analysis and Recovery Validated PAR Immunoassay
Human Tumor Extract Titration
0
100
200
300
400
500
600
700
800
900
0 2 4 6 8 10 12
Tumor lysate (μg)
PA
R (p
g/m
L)
D2
D3
D4
D5
D6
D7
D8
D9
D10
D15
B4
B7
Spiked Read = f(Std Curve)
0.E+00
1.E+05
2.E+05
3.E+05
4.E+05
0.E+00 1.E+05 2.E+05 3.E+05 4.E+05 5.E+05 6.E+05
PAR Standard Curve RLU
PA
R s
pike
d in
to e
xtra
ct, R
LU
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Assay Validation
• Most Critical Aspect: Demonstrate that the assay is fit for purpose
– An assay with a 20% interday/instrument/operator CV cannot detect a 20% modulation of signal
– Must know accuracy and precision to assess performance
– This does not capture biological variation, which is large
• Analytical sensitivity yields real-world benefits:
– Allows repeat determinations on the same specimen
– Adapted for the size of the specimen to be used in the clinic: e.g., an 18-gauge needle biopsy
• Robustness: Must be transferable to other labs
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PAR Immunoassay Development
• Key changes made included:– Specimen treatment step added– Specimen diluent and dilution
level– Specimen incubation time and
temperature– Substrate incubation time and
temperature• Other Changes
– Use of a shaking step before the read
– Blockers– Carrier protein – Read time – Instrument settings– Conjugate, conjugate
concentration, and diluent
[PAR] Total %CV
Previous New
10 46.4 ? 30 30.2 8.1 100 24.6 7.4 300 19.5 6.9 1000 13.9 6.4
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Completion of a Validated Assay Format
• Selection of assay format • Production of assay controls (QC samples)• Adequate instrumentation (calibrated and validated)• Selection of standards and the standards matrix
– Will the standards matrix be a cell extract?– How will that affect stability of the standards?
• Adequate assay sensitivity for multiple determinations on the same clinical specimen
• Specific optimization of handling by specimen type • Validation issues:
– Use common reagents (master lot)– Precision validation (operator, day, instrument)– Accuracy validation (dilution linearity, spike recovery)– Assay transfer between sites
NOW WE CAN START!
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Now Start Application in Preclinical Modeling
• Establish the dynamic range of response to treatment
• Time window of sampling consistent with clinical realities
• Tie the dose/response curve in tumor growth inhibition to marker modulation
– What is the minimum detectable signal?
– Is that in the microdose range?
– Can the assay report whether an effective dose is delivered to the tumor?
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Vehicle Control Biopsies at 100x, All 4 Mice
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Intratumoral γ-H2AX Response to 4.7 mg/kg Topotecan +2 Hours, Mouse #22, All 3 Fields
The biopsy assay counts pixels to determine the fraction of nuclear area that is γ-H2AX-positive over three 200x fields. It uses the DAPI signal toidentify fields containing mostly live cells.
It matters where the needle goes!
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Comparison of H2AX Response to Topotecan or Indenoisoquinoline Drug Dose, +2-Hour Timepoint
H2AX Dose Response
-0.5
0
0.5
1
1.5
2
2.5
-1 0 1 2 3 4 5
Ln Dose (mg/kg)
Ln
%N
AP
-V
TPT
724998725776
706744
• Plot is vehicle- corrected Ln/Ln with plateau response points omitted for topotecan (TPT) and NSC 725776
• Range of vehicle reads was the same across the 3 experiments; means & SDs overlapped
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Helpful Links and References
• http://www.westgard.com/lesson.htm
• http://www.nihs.go.jp/drug/validation/q2bwww.html
• http://www.fda.gov/cder/guidance/4252fnl.htm
• http://www.clinchem.org/info_ar/anal_meth.shtml
• http://www.cbrlabs-inc.com/assay-validation.html
• Validation of Immunoassays for Bioanalysis: a Pharmaceutical Industry Perspective. Findlay J., Smith W., Lee D., Nordblom G., Das I., DeSilva B., Khan M., and Bowsher R. Journal of Pharmaceutical and Biomedical Analysis. 2000; 21: 1249-1273.
• Immunoassay, A Practical Guide. Ed. Dan Chan and Marie Pearlstein. Academic Press, 1987, 1992.
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Operations and Technical Support Contractor for
The National Cancer Institute at Frederick
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The Next Speaker is:
Dr. Susan Galbraith