Abstract 818: Correlation of mRNA and protein expression of CA125 and HE4 in ovarian cancer tissues using real-time RT-PCR and reverse phase protein array technologies
Authors: Murphy Patricia , Adams Melissa , Martin Brennan , Horlick Elizabeth , Malinowski Douglas 
Published: February 2011

Abstract
BACKGROUND: Epithelial ovarian cancer is the fifth most frequent cause of cancer deaths among women in the United States. Prognosis is improved when it is detected in early stages but poor as the disease progresses. Monitoring for ovarian cancer currently uses the serum marker CA125, although it is not very effective in detecting early stage ovarian cancers. Recently, the biomarker HE4 has shown clinical utility in the detection of ovarian cancer. However, it is likely that additional markers are needed to detect early stage ovarian cancer.
OBJECTIVE: This study investigated whether reverse phase protein array (RPPA) technology would complement real time RT-PCR and serve as an additional tool to screen for biomarkers. RPPA is a sensitive, high throughput, quantitative assay that measures small amounts of proteins simultaneously on a single slide.
METHODS: The mRNA expression levels of CA125 and HE4 were measured using real-time quantitative RT-PCR from 27 normal ovary tissue specimens and 54 epithelial ovarian tumors. The protein levels for these biomarkers were assessed in tissue lysates from the same normal ovary and ovarian tumor specimens by immunoblotting and reverse phase protein arrays.
RESULTS: CA125 mRNA was elevated in 76% (41/54) of the tumors at levels greater than 10 fold relative to the average expression in normal ovary tissue. Fifty-nine percent (32/54) of epithelial tumors expressed CA125 mRNA at greater than 100 fold compared to normal ovarian tissue. HE4 mRNA was over expressed in 84% (42/50) of the tumors at levels 10 fold greater and in 68% of the tumors at 100 fold greater than the average normal ovary tissue. Protein expression levels of the CA125 and HE4 biomarkers were measured by immunostaining tissue lysates on RPPAs. As observed for CA125 and HE4 mRNAs, there were statistically significant differences in the protein levels detected between normal ovary and ovarian tumor for both biomarkers. CA125 protein was detected in 81% of tumors at 2.5 fold greater than, and in 75% of the tumors at 5 fold greater than, the average of the normal ovary tissue. Seventy-seven percent of tumors had HE4 protein detected at levels greater than 2.5 fold and 30% greater than 5 fold compared to normal ovary tissue. Immunoblotting validated the RPPA results for HE4 protein expression in a subset of the tissue lysates. Importantly, a significant correspondence between the mRNA and protein levels of each biomarker was demonstrated with correlation coefficients of 0.88 and 0.78 for CA125 and HE4, respectively.
CONCLUSION: In this dataset, there is a robust correlation between mRNA and protein levels for HE4 and CA125. As RPPA yielded similar results compared to immunoblotting, is quantitative, and has higher throughput, RPPA is a valuable method for high throughput analysis of protein expression in tissue lysates.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 818.