Objective quantitation of EGFR protein levels using Quantitative Dot Blot (QDB) method for prognosis of gastric cancer patients

Abstract

Background: One causing factor underlying failures of several clinical trials of anti-EGFR therapies is the lack of effective method to select patients overexpressing EGFR protein. Quantitative Dot Blot method (QDB) is proposed here to measure EGFR protein levels objectively and quantitatively. Its feasibility was evaluated for prognosis of overall survival (OS) of gastric cancer patients. Methods: FFPE slices of 2X5 microM from gastric and Lung cancer specimens were used to extract total tissue lysates for QDB measurement. Absolutely quantitated EGFR protein levels were used for Kaplan-Meier Overall Survival (OS) analysis of gastric cancer patients. Results: EGFR protein levels ranged from 0 to 772 pmole/g for gastric cancer specimens (n=246), and from 0 to 2695 pmole/g for lung cancer patients (n=81). Poor correlation was observed between quantitated EGFR levels and IHC scores with r=0.018, p=0.786 from Spearman correlation analysis. EGFR was identified as an independent negative prognostic biomarker for gastric patients only through absolute quantitation, with HR at 2.29 (95%CI:1.23-4.26, p=0.0089) from multivariate cox regression OS analysis. A cutoff of 207.7 pmole/g was proposed to stratify gastric cancer patients, with 5-year survival probability at 37% for those whose EGFR levels were above the cutoff, and at 64% those below the cutoff based on Kaplan-Meier OS analysis. p=0.0057 from Log Rank test. Conclusion: A QDB-based assay was developed for both gastric and Lung cancer specimens to measure EGFR protein levels absolutely, quantitatively and objectively. This assay should facilitate clinical trials aiming to evaluate anti-EGFR therapies retrospectively and prospectively.