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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
Published on: June 6, 2022
Protocol modifications influence the result of EGF receptor immunodetection by EGFR pharmDx in paraffin-embedded
Katalin Derecskei1, Judit Moldvay, Krisztina Bogos
1Department of Tumor Progression, National Institute of Oncology, Budapest, H-1122, Hungary. jtimar@oncol.hu.
Abstract:
EGF receptor (EGFR) became a useful target for several recently introduced therapies of various cancer types including colorectal, lung, head and neck cancers and glioblastoma. The successful clinical application of these novel molecularly targeted therapies requires the expression of their target, EGFR, determined by nucleic acid based or immunohistochemical techniques. However, until now, immunohistochemistry has not become a reliable diagnostic approach for this purpose. The golden standard for the determination of EGFR protein expression in paraffin-embedded cancer tissues is the EGFR pharmDxTM kit. Here we show that the recommended protocol may not be optimal for EGFR immunodetection. Microwave antigen retrieval and extended primary antibody incubation time converted four out of eight EGFR-negative tumors into EGFR-positive in a study of 50 lung adenocarcinoma cases. Accordingly, we recommend retesting cases negative for EGFR with EGFR pharmDxTM using protocol modifications optimizing antigen retrieval and the incubation periods.
Insights
Optimizing the EGFR pharmDxTM kit protocol can improve diagnostic accuracy. Modified antigen retrieval and incubation times identified EGFR in previously negative lung adenocarcinoma cases, suggesting retesting is needed.
Area of Science:
- Oncology
- Molecular Diagnostics
- Cancer Research
Background:
- Epidermal Growth Factor Receptor (EGFR) is a key target for molecularly targeted therapies in various cancers.
- Accurate determination of EGFR expression is crucial for selecting patients for these treatments.
- Current immunohistochemistry (IHC) methods for EGFR detection, including the EGFR pharmDxTM kit, may lack optimal reliability.
Purpose of the Study:
- To evaluate the reliability of the standard EGFR pharmDxTM kit protocol for EGFR immunodetection.
- To investigate the impact of modified antigen retrieval and antibody incubation on EGFR detection in lung adenocarcinoma.
- To propose optimized protocols for more accurate EGFR expression assessment.
Main Methods:
- Analysis of 50 lung adenocarcinoma cases.
- Application of the standard EGFR pharmDxTM kit protocol.
- Modification of the protocol including microwave antigen retrieval and extended primary antibody incubation time.
Main Results:
- The standard EGFR pharmDxTM protocol classified four out of eight initially EGFR-negative tumors as EGFR-positive after protocol modifications.
- Optimized antigen retrieval and extended incubation periods significantly altered EGFR detection results.
- These modifications converted previously negative cases to positive, highlighting potential underdiagnosis with standard methods.
Conclusions:
- The recommended protocol for the EGFR pharmDxTM kit may not be optimal for EGFR immunodetection.
- Modifications in microwave antigen retrieval and primary antibody incubation can enhance EGFR detection sensitivity.
- Retesting of EGFR-negative cases using optimized protocols is recommended for improved diagnostic accuracy in lung adenocarcinoma.
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