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 Approach

Asterand Bioscience 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

 

cDX-TMA

cDXTMA_Web_Image

Cell lines: FFPE-cores containing gastric, breast and colorectal cancers (known MET levels)
Gastric Cancers: FFPE-tissue cores from five randomly selected cases
Rc-MET: FFPE tissue matrices spiked with recombinant human MET (12 concentrations)

 

Quantitative IHC

Aperio Digital scanning and image analysis allows determination of specificity, range and linearity.

Aperio_web_Image2

 

Quantitative assessment of the performance of two anti-MET antibodies (BLUE and RED lines) at a single assay condition (pH9 HIER, 0.5mg/ml – Dako Envision Flexplus), against that of a non-immune control (GREEN) over twelve concentrations of recombinant MET.

 

For further information, or to discuss a specific study, contact us at advantage@asterandbio.com.