Validation of new aromatase monoclonal antibodies for immunohistochemistry: progress report

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Abstract

Intratumoral aromatase is a potential therapeutic target for the treatment of postmenopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. A multi-center collaborative group has been established to generate and validate new aromatase monoclonal antibodies (MAbs). A recombinant GST–aromatase fusion protein was expressed in baculovirus and the purified protein was used for immunization of mice either as a native or formalin-fixed antigen. Hybridomas were generated using standard techniques and screened biochemically prior to immunohistochemistry (IHC) evaluation in human placenta, ovary and breast cancer tissues. Twenty-three MAbs selected by biochemical assays were further evaluated by IHC of paraffin-embedded tissue sections including normal ovary, and placenta, and a small series of 10 breast carcinomas. Of the 23 MAbs, 2 (clones 677 and F2) were determined to specifically stain cell types known to express aromatase in normal tissues. In breast carcinomas staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments was detected. IHC was performed and independently evaluated by three pathologists (HS, TJA and SGS), each using the same evaluation criteria for staining intensity and proportion of immunopositive cells. With these two MAbs, interpathologist and intralaboratory variations were minimal in comparison with differences which could be detected between tissue specimens and antibodies.

Introduction

Estrogens are considered to play important roles in the development and progression of hormone-dependent human breast cancer. Human breast cancers express increased aromatase enzyme and activity compared with normal breast tissue. The overexpression of aromatase appears to play an important role in estrogen related development and progression of some human breast cancers [1], [2], [3], [4]. Aromatase inhibitor therapy is one of the endocrine treatments available to breast cancer patients. It has therefore become very important to predict which patients will respond prior to initiation of therapy.

At present, aromatase inhibitors appear to exert their effects primarily through the reduction of aromatase activity in non-ovarian tissue in postmenopausal women. Some studies have demonstrated a positive correlation between intratumoral aromatase activity and response to treatment with various aromatase inhibitors [3], [4]. If in situ estrogen biosynthesis and local concentrations are important in the cancer cases that respond to aromatase inhibitor treatment, the tumors must also express the aromatase enzyme as well as estrogen and progesterone receptors. Accordingly, measurement of aromatase activity might serve as a useful tool for prediction of responses to aromatase inhibitors. Test of this concept has been limited by the need to determine aromatase by biochemical methods in fresh tissue. What is needed is a precise, sensitive, and quantifiable method for detecting aromatase in archival materials or formalin-fixed and paraffin-embedded tissue. No anti-aromatase antibodies have been specifically designed for this purpose.

Existing antibodies directed against aromatase are available. However, they are in ever dwindling supplies and the results from studies using them have been controversial in terms of tumor aromatase localization [5], [6], [7], [8], [9]. Therefore, this study was undertaken as an international collaboration in order to develop aromatase antibodies that can be used to assess aromatase expression in fixed breast cancer tissue and test whether these measurements are predictive of responsiveness to aromatase inhibitors. This manuscript represents an interim progress report of this project.

Section snippets

Materials

Tissues were fixed in 3.7% formalin and embedded in paraffin. Control tissues included normal placenta and cycling ovary. All human breast carcinoma (10 cases) used for initial screening were invasive ductal carcinoma and retrieved from surgical pathology files of Department of Pathology, Tohoku University Hospital.

Production of monoclonal antibodies (MAbs) against aromatase

Preparation of aromatase antigen, its fixation prior to injection, initial screening for antibody production, and development of hybridomas were carried out in a fashion similar to

Selection criteria for MAbs to aromatase and results of hybridoma screening

The strategy for screening hybridomas was to assay the initial fusion wells by ELISA against purified GST–aromatase used as antigen and with free GST. Only positives for GST–aromatase and negatives for the GST moiety of the antigen were selected. ELISA positives were further screened by Western blot against purified GST–aromatase, GST–aromatase in crude extracts of Sf9 cells and free GST. Hybridoma products that gave a specific reaction for GST–aromatase by Western blot with little or no

Discussion

Due to marked improvements in immunostaining methods and the antibodies used, it is now possible to immunolocalize the increasing number of antigens in routinely processed specimens (i.e. 10% formalin-fixed and paraffin-embedded tissue). Immunohistochemistry can now be performed rapidly and without many technical difficulties. We reasoned that antibodies against aromatase and an immunohistochemical staining system would allow semiquantitation of aromatase immunoreactivity in tissue sections in

Acknowledgements

The authors acknowledge the technical assistance of Kurt Christensen and Lori Sherman (UCHSC).

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Presented at the VI International Aromatase Conference: AROMATASE 2002, Kyoto, Japan, 26–30 October 2002.

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