Cytometry Volume 35  Issue 1  1999 Pages: 2-10

Abstract Introduction Methods Results Discussion

Abstract

Background:

The immunostaining features of the androgen receptor (AR) have been studied in prostate cancer (CaP) to predict the outcome of androgen deprivation therapies. We have developed an automatic video color image analysis system for quantitation of AR expression in large samples of prostatic nuclei.

Methods:

Essential criteria of immunostaining have been examined to establish a linear relationship between AR protein content and mean optical density (MOD) of the immunoperoxidase-substrate reaction product. Titration of monoclonal AR antibody, F39.4.1, and concentration and reaction time of substrate were optimized using color video image analysis. The methodology was tested twice. First, CWR22 human CaP xenograft specimens, harvested from testosterone (T)-stimulated, castrated and T-resupplemented mice, were immunostained to demonstrate the dependence of AR expression on serum androgen levels. Second, AR expression was measured in archived clinical specimens.

Results:

In CWR22 tumor-bearing mice castrated for 6 days, AR MOD decreased to 57% of T-stimulated, intact mice. After 72 hrs of T treatment, AR MOD returned to the level measured in T-stimulated, intact mice. Sixteen radical prostatectomy specimens and 16 transurethral resection of prostate (TURP) specimens were double-labeled with F39.4.1 and anti-cytokeratin MAb (13E12) specific for basal epithelial cells. Benign epithelial cells exhibited lower AR MOD in prostatectomy compared to TURP specimens (P < 0.01). Differences in AR immunostaining intensity may have resulted from differences in tissue fixation of whole organ versus small tissue specimens.

Conclusions:

AR immunostaining can be quantitated accurately using optimized immunohistochemical criteria and video image analysis. Cytometry 35:2-10, 1999.  1999 Wiley-Liss, Inc.

^*Correspondence to James L. Mohler, UNC-Lineberger Cancer Center, CB# 7295, University of North Carolina, Chapel Hill, NC 27599-7295.

Funding Agency: National Institutes of Health; Grant Number: AG11343 (to J.L.M.), CA64865 (to D.K., J.L.M., and G.J.S.), P30-HD-18968 (DNA and tissue culture cores)
Funding Agency: American Foundation for Urologic Disease (to C.W.G.)
Funding Agency: Merck U.S. Human Health (to C.W.G.)
 
 

Methods and materials

Color Video Image Processing
 

Color image in RGB color scheme	Local adaptive thresholding [Ref. 23] and logical OR of RGB images	Classification of positive cells	Artifacts removed and mean optical density of cell nuclei assigned

 

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