Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative...

Area of Science:

• Biochemistry
• Molecular Biology
• Clinical Diagnostics

Background:

• Accurate protein biomarker quantitation in Formalin Fixed Paraffin Embedded (FFPE) samples is crucial for clinical diagnostics.
• Existing methods may lack objectivity, consistency, and accuracy in daily practice.

Purpose of the Study:

• To explore the feasibility of the Quantitative Dot Blot (QDB) method for absolute protein biomarker quantitation in FFPE samples.
• To establish QDB as a reliable immunoassay for clinical applications.

Main Methods:

• Extraction of total protein lysate from FFPE tissue slices.
• Absolute and quantitative measurement of HER2 protein levels using the QDB method.
• Correlation analysis with immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) data.

Main Results:

• High correlation (r=0.963) between HER2 levels measured by QDB and IHC using validated antibodies.
• Achieved high Area Under the Curve (AUC) values (0.9998 ± 0.0002 for IHC, 0.9942 ± 0.0031 for IHC+FISH).
• Demonstrated high concordance (99.5% with IHC, 96.9% with IHC+FISH) after dichotomous conversion of results.

Conclusions:

• The QDB method is the first immunoassay platform capable of absolute protein quantitation in FFPE samples.
• QDB meets the needs of daily clinical practice, particularly for local or developing country laboratories.
• This method enhances objectivity, consistency, and accuracy in protein biomarker analysis.