Objective Quantitation of EGFR Protein Levels using Quantitative Dot Blot Method for the Prognosis of Gastric Cancer Patients

Abstract

Purpose An underlying factor for the failure of several clinical trials of anti-epidermal growth factor receptor (EGFR) therapies is the lack of an effective method to identify patients who overexpress EGFR protein. The quantitative dot blot method (QDB) was used to measure EGFR protein levels objectively, absolutely, and quantitatively. Its feasibility was evaluated for the prognosis of overall survival (OS) of patients with gastric cancer. Materials and Methods Slices of 2×5 μm from formalin-fixed paraffin-embedded gastric cancer specimens were used to extract total tissue lysates for QDB measurement. Absolutely quantitated EGFR protein levels were used for the Kaplan-Meier OS analysis. Results EGFR protein levels ranged from 0 to 772.6 pmol/g (n=246) for all gastric cancer patients. A poor correlation was observed between quantitated EGFR levels and immunohistochemistry scores with ρ=0.024 and P=0.717 in Spearman's correlation analysis. EGFR was identified as an independent negative prognostic biomarker for gastric cancer patients only through absolute quantitation, with a hazard ratio of 1.92 (95% confidence interval, 1.05–3.53; P=0.034) in multivariate Cox regression OS analysis. A cutoff of 208 pmol/g was proposed to stratify patients with a 3-year survival probability of 44% for patients with EGFR levels above the cutoff versus 68% for those below the cutoff based on Kaplan-Meier OS analysis (log rank test, P=0.002). Conclusions A QDB-based assay was developed for gastric cancer specimens to measure EGFR protein levels absolutely, quantitatively, and objectively. This assay should facilitate clinical trials aimed at evaluation of anti-EGFR therapies retrospectively and prospectively for gastric cancer.

ELISA-like QDB method to meet the emerging need of Her2 assessment for breast cancer patients

“…The QDB method shares a lot of features with ELISA, including being high throughput, objective, quantitative, and easy to perform. Yet, unlike ELISA, this method is able to measure antigen levels in FFPE specimens (20)(21)(22)(23). Thus, we considered it as an ELISA-like assay.…”

Validation of absolutely quantitated Ki67 and cyclinD1 protein levels for prognosis of Luminal‐like breast cancer patients

“…Recently, Quantitative Dot Blot (QDB) method was developed to measure a broad range of protein biomarkers as absolute and continuous variables 11–15 . This method was used to measure Ki67 and cyclinD1 levels absolutely and quantitatively in a cohort of 155 Formalin Fixed Paraffin Embedded (FFPE) luminal‐like breast cancer specimens (Cohort 1) 16,17 .…”

“…4,5 Recently, Quantitative Dot Blot (QDB) method was developed to measure a broad range of protein biomarkers as absolute and continuous variables. [11][12][13][14][15] This method was used to measure Ki67 and cyclinD1 levels absolutely and quantitatively in a cohort of 155 Formalin Fixed Paraffin Embedded (FFPE) luminal-like breast cancer specimens (Cohort 1). 16,17 The absolutely quantitated Ki67 levels were used to replace IHC-based Ki67 scores in surrogate assay, and the patients were stratified using an outcome-based cutoff of 2.31 nmol/g into Luminal A-like (LumA q ) and B-like subtype (LumB q ).…”