Detection of progesterone receptor forms A and B by immunohistochemical analysis.
OBJECTIVE
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.
Mote
P A
PA
Westmead Institute for Cancer Research, University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia. patricia_mote@mail.wmi.usyd.edu.au
Johnston
J F
JF
Manninen
T
T
Tuohimaa
P
P
Clarke
C L
CL
D000818:Animals; D000906:Antibodies; D001943:Breast Neoplasms / Q000737:chemistry; D019556:COS Cells; D018270:Carcinoma, Ductal, Breast / Q000737:chemistry; D004717:Endometrium / Q000737:chemistry; D005260:Female; D005557:Formaldehyde; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D011817:Rabbits; D011980:Receptors, Progesterone / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000737:chemistry
D000906:Antibodies; D011980:Receptors, Progesterone; C101118:progesterone receptor A; C097314:progesterone receptor B; D005557:Formaldehyde