Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.

Lung squamous cell carcinoma (LSCC) has a high mutational burden and poor prognosis, even with immunotherapy. In the Lux-Lung 8 trial, afatinib, an epidermal growth factor receptor (EGFR) inhibitor, showed a long-term benefit in 5.3% of patients with LSCC. Because activating mutations of EGFR are rare in LSCC, the response was likely due to wild-type EGFR being activated by an unknown mechanism. All receptor tyrosine kinase (RTK) proteins, both wild-type and mutated, are activated by phosphorylation of specific tyrosines which serve as binding sites for various SH2 proteins. The aim of this feasibility study was to determine whether enhanced chemiluminescent western blotting (WB) with a phosphotyrosine (pTyr) antibody is sufficiently sensitive to detect pTyr-RTK proteins in human LSCC tissues. We performed WB analysis on 25 resected human lung tissue samples, including 12 LSCC, two adenocarcinomas (LADC), and 11 control (non-tumor) lung samples. The analysis showed ~220 kDa pTyr-protein bands in two LSCC samples that were much more abundant than the corresponding bands in controls or LADC samples. To identify pTyr-RTKs, pTyr WB patterns of the two samples were compared to those of five RTK candidates: EGFR, platelet-derived growth factor receptor beta (PDGFRB), vascular endothelial growth factor receptor, anaplastic lymphoma receptor, and mesenchymal-epithelial transition factor. The strong pTyr signal in one sample matched EGFR, whereas the other matched a combination of EGFR and PDGFRB. We conclude that pTyr WB is sufficiently sensitive to detect pTyr-RTK drivers in flash-frozen tumor tissues and might identify LSCC patient subsets responsive to RTK inhibitors.