Validation of IHC assays as Companion Diagnostics (cDX)
With increasing use of biomarkers to guide therapeutic decisions, aid stratification of patients into treatment groups and evaluate experimental therapeutics, the reliance on validated assays has become paramount.
Our PHASEZERO Research team has developed an efficient and effective approach to the validation of IHC assays to the standards required by regulatory authorities:
- Bioanalytical Method Validation: CDER and CVM 05/2001.
- Guidance for Submission of Immunohistochemistry Applications to the FDA: CDRHN 06/1998.
- Q2B Validation of Analytical Procedures: Methodology: CDER and CBER November 1996
These documents define the steps required to demonstrate the specificity and overall reliability of the assays developed.
Case Study – MET
c-Met (MET, hepatocyte growth factor receptor) is a receptor tyrosine kinase expressed in epithelial cells in many normal tissues. MET is de-regulated in a wide range of human cancers and based on its importance to oncology research the target was selected for the current project, which aims to develop and validate an IHC assay for MET in human tissues.
We performed a series of experiments to address each of the primary validation criteria as defined by the regulatory documents, namely:
SPECIFICITY, LINEARITY, RANGE, ACCURACY, PRECISION and ROBUSTNESS
Our approach utilizes a rigorous but highly efficient experimental design and multiple complementary technologies, including digital pathology and quantitative image analysis.
SPECIFICITY: Antibody selection (3): western blot / IHC in cell lines
LINEARITY / RANGE: Quantitative IHC analysis of a custom-made tissue microarray (‘cDX-TMA’) (illustrative data shown below)
ACCURACY: Analysis of multiple antibodies IHC analysis cDX-TMA
PRECISION: Sub-cellular localization and assay reproducibility inter / intra-laboratories
ROBUSTNESS: Quantitative IHC analysis of cDX-TMA over range of assay variables
Aperio Digital scanning and image analysis allows determination of specificity, range and linearity.