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 KaplanMeier 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.
ABSTRACT
Objective To construct the fusion gene of membrane type matrix metalloproteinase1 humanized tandem affinity purification (MT1 MMP hTAP) and to evaluate the expression of its protein complexes isolated and purified using tandem affinity purification (TAP) system. Methods The MT1 MMP hTAP protein complexes were isolated and purified using the IgG agarose beads and calmodulin sepharose beads affinity chromatography according to the molecular and biological features of TAP system. The expression levels of its protein complexes were determined by PAP immunostaining and Western blotting. Results PAP antibody immunostaining showed that MT1 MMP hTAP protein complexes (dark blue) were expressed in chick fibroblast cells. Western blot analysis of its protein complexes using MT1 MMP specific antibody showed a prominent band (68?10 3) of mobility consistent with fully processed MT1 MMP hTAP after being cleaved by the TEV proteinase. Conclusion The construction of TAP system in chick fibroblast cells and expression of MT1 MMP hTAP protein complexes will be helpful for the studies of the MT1 MMP hTAP interaction proteins in the human tumor cells.
