Epidermal growth factor receptor in breast cancer: Storage conditions affecting measurement, and relationship to steroid receptors

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Abstract
This study investigates the effect of freezing and storage of tissue and subcellular fractions on the measurement of epidermal growth factor receptors (EGF-r); compares competition binding and single saturating dose assays (SSD) for quantitating EGF-r levels; investigates several tissues as potential quality control; and examines the relationship between EGF-r and hormone receptor expression in human breast cancers. Mouse and calf uterine cell membranes were preferred sources of quality control tissue with similar levels of high affinity EGF-r to human breast cancer tissue (<150–200 fmol/mg membrane protein). Studies using pooled mouse uterine tissues indicated a loss of 40% in EGF-r activity following a single −20°C freeze/thaw cycle, while a breast cancer tissue showed a 75% loss, independent of storage temperature (liquid nitrogen, −70°C, −20°C). A single freeze/thaw cycle of mouse uterine broken cell pellets (nuclei plus membrane fraction) again indicated a loss of EGF-r irrespective of storage temperature (43% loss at −70°C, 52% loss at −20°C). In most cases irrespective of the tissue type or tissue fraction being stored, the length of storage had little impact on the extent of the loss in activity. A second freeze/thaw cycle of intact tissue, or freezing of broken cell pellets from a previously-frozen tissue, led to a further major or total loss of the remaining EGF-r. Overall these results are commensurate with the published effects of freezing and storage on estrogen receptor measurement. In addition, our studies suggest that the most suitable procedure for assaying frozen breast cancer specimens for EGF-r levels in conjunction with steroid receptor quantitation is to prepare and assay both cytosol and membrane fractions for their respective receptor content without further storage. A concordance of 86% was found in 44 breast cancers assayed for EGF-r by saturation analysis and SSD. Statistically significant inverse relationships were found between EGF-r and estrogen and progesterone receptor levels in a study of approximately 350 breast cancer patients. No association was found with tumor stage or diameter, axillary node involvement, or patient age.

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