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Purpose: We have applied the sensitive and specific in situ proximity ligation assay (PLA) to characterize Tyr1068 phosphorylation of the epidermal growth factor receptor (EGFR) in cervical cancer in relation to the protein level and gene dosage., Materials and Methods: Pretreatment tumor biopsies from 178 patients were analyzed. EGFR protein level was determined by immunohistochemistry, and Tyr1068 phosphorylation was detected with PLA in 97 EGFR positive tumors. EGFR gene dosage was derived from array comparative genomic hybridization of 86 cases., Results: EGFR was expressed in most tumors, whereas phosphorylation was seen in about half of the EGFR positive ones. A correlation was found between the expression of EGFR and phosphorylated EGFR (p=0.016, membrane; p=0.012, cytoplasm). However, tumor regions with high protein level without phosphorylation were occasionally seen and the percentage of EGFR positive cells was higher than the phosphorylated percentage (p<0.001). Moreover, an increase in the phosphorylation in both the membrane (p=0.014) and cytoplasm (p=0.002) was seen in 11 tumors with gain of EGFR. The protein level was not correlated with gene dosage., Conclusion: In contrast to gain of the EGFR chromosomal region, high EGFR protein level may not necessarily indicate Tyr1068 phosphorylation and thereby receptor activation in cervical cancer., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Purpose: We compared the prognostic significance of ectodomain isoforms of the epidermal growth factor receptor (EGFR), which lack the tyrosine kinase (TK) domain, with that of the full-length receptor and its autophosphorylation status in cervical cancers treated with conventional chemoradiotherapy., Experimental Design: Expression of EGFR isoforms was assessed by immunohistochemistry in a prospectively collected cohort of 178 patients with squamous cell cervical carcinoma, and their detection was confirmed with Western blotting and reverse transcriptase PCR. A proximity ligation immunohistochemistry assay was used to assess EGFR-specific autophosphorylation. Pathways associated with the expression of ectodomain isoforms were studied by gene expression analysis with Illumina beadarrays in 110 patients and validated in an independent cohort of 41 patients., Results: Membranous expression of ectodomain isoforms alone, without the coexpression of the full-length receptor, showed correlations to poor clinical outcome that were highly significant for lymph node-negative patients (locoregional control, P = 0.0002; progression-free survival, P < 0.0001; disease-specific survival, P = 0.005 in the log-rank test) and independent of clinical variables. The ectodomain isoforms were primarily 60-kD products of alternative EGFR transcripts. Their membranous expression correlated with transcriptional regulation of oncogenic pathways including activation of MYC and MAX, which was significantly associated with poor outcome. This aggressive phenotype of ectodomain EGFR expressing tumors was confirmed in the independent cohort. Neither total nor full-length EGFR protein level, or autophosphorylation status, showed prognostic significance., Conclusion: Membranous expression of ectodomain EGFR isoforms, and not TK activation, predicts poor outcome after chemoradiotherapy for patients with lymph node-negative cervical cancer., (©2011 AACR.)