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. 2001 Aug;54(8):624–630. doi: 10.1136/jcp.54.8.624

Detection of progesterone receptor forms A and B by immunohistochemical analysis

P Mote 1, J Johnston 1, T Manninen 1, P Tuohimaa 1, C Clarke 1
PMCID: PMC1731503  PMID: 11477119

Abstract

Aim—The measurement of progesterone receptors (PR) is recommended as part of the clinical management of breast and endometrial cancers, and immunohistochemistry on formalin fixed tissue is now the method of choice. PR is expressed as two isoforms, PRA and PRB, and although both these proteins are expressed in hormone dependent cancers, there is evidence that a large proportion of tumours express a predominance of one isoform. Therefore, it is essential to document the individual detection of PRA and PRB by the presently available anti-PR antibodies. The aim of this study is to investigate the detection of PR isoforms A and B in formalin fixed, paraffin wax embedded cell lines and tissue sections by immunohistochemistry, using a panel of commercial and in house antibodies to human PR.

Methods—PR negative cell lines stably transfected to express only PRA (MCF-7Mll/PRA) or PRB (MDA-MB-231/PRB), and tissue sections of human breast carcinoma and normal endometrium were stained using an immunoperoxidase method. A panel of primary PR specific antibodies was evaluated for ability to detect both PRA and PRB proteins, and for intensity and distribution of positive staining under optimal conditions.

Results—Of the 11 antibodies assessed, only four recognised PRA and PRB similarly. Six recognised PRA proteins but were unable to detect PRB expression in the cell lines expressing only PRA or PRB. In tissues expressing high amounts of PRA and PRB, all antibodies tested demonstrated positive PR staining. However, in tissues expressing a predominance of PRB, differential staining patterns were observed, with variations in staining intensity and in the proportion of cells positive for PR.

Conclusions—Most PR specific antibodies tested failed to detect PRB in formalin fixed tissue by immunohistochemical techniques, despite their ability to do so by immunoblot analysis. These observations suggest that there are conformational differences between PRA and PRB that mask epitopes on the PRB protein recognised by most anti-PR antibodies. The selection of antibodies that recognise both PRB and PRA in formalin fixed tissue is essential for the accurate evaluation of PR positivity in clinical specimens.

Key Words: progesterone receptor isoforms • progesterone receptor antibodies • immunohistochemistry

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Figure 1 Immunoperoxidase staining of progesterone receptor isoform A and B (PRA and PRB) proteins in a PR negative cell line transfected with PRA (MCF-7M11/PRA) (A, C, E, and G); a PR negative cell line transfected with PRB (MDA-MB-231/PRB) (B, D, F, and H); or midsecretory endometrium (I–L). The primary antibodies used are:- Cell Marque (A, B, and I); PR-86 (C, D, and J); Novocastra (E, F, and K); and hPRa6 (G, H, and L). Original magnification, x400.

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Figure 2 Immunoblot analysis of human progesterone receptor isoforms A and B (PRA and PRB) with PR-86. Equal amounts of total protein from COS cells transfected with human PRA and PRB were analysed by immunoblot using PR-86. Lane 1, negative control (transfected with vector only); lane 2, COS cells transfected with PRB; lane 3, COS cells transfected with PRA.

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