We write to update our previous report (1) of immunohistochemical analysis of frozen breast cancer samples for the hENT11 protein. In this paper, we analyzed 33 samples of frozen breast cancers and identified marked variability in the staining intensities for hENT1 protein, normalized to the staining of the internal positive control provided by endothelial cell membranes. We have refined and validated this immunohistochemical analysis for assessment of formalin-fixed paraffin-embedded tissues, as described below.
Twenty-two formalin-fixed, paraffin-embedded breast tumors were obtained from the previously reported series of 33 frozen breast tumors. Paraffin sections of 4–6-μm thick were dried in an oven at 59°C for 2 h. Sections were brought down to water through three changes of xylene (10 min each), then through graded alcohol from 100% to 50%, and then water. Slides were microwaved in TT-Mega (milestone) under control temperature and high pressure for 2 min at 100°C in 1 mm EDTA (pH 8.0), cooled for 6 min, and washed in running cold tap water. Slides were incubated in 10% H2O2 in methanol and rewashed for 5 min in tap water. Tissue sections were incubated with the anti-hENT1 monoclonal antibodies used for the previous study at room temperature in a humidified chamber for 30 min. The section was then rinsed in PBS (pH 7.2), immersed in buffer for 5 min, incubated with Envision (DAKO EnVision+) goat antimouse dextran conjugate for 30 min, washed in PBS for 5 min, incubated with diaminobenzidine solution, rinsed, counterstained with hematoxylin, dehydrated through graded alcohols and xylene, and coverslipped. Negative controls were provided by omitting the primary antibodies, by hENT1 peptide-eluted antibody preparations, and by substitution of primary antibodies with anti-CD-138 monoclonal antibodies. Immunohistochemistry was assessed and scored subjectively by a single pathologist (R. W. C.) blinded to the original hENT1 staining intensities reported for the frozen samples.
Endothelial cell plasma membrane staining was observed and assigned a score of 2+. The membrane staining of invasive adenocarcinoma cells was graded in reference to endothelial cell staining, with weaker but definite positive staining given a score of 1+, the most intense staining given a score of 4+, and staining intermediate between 4+ and 2+ (endothelial cells) given a score of 3+ (see Fig. 1). Adenocarcinoma cell membrane staining was absent in five samples, 1+ in two samples, 2+ in eight samples, 3+ in six samples, and 4+ in a single sample. The paraffin staining intensities of these 22 samples were then analyzed for the strength of association with the paired frozen staining intensities using Pearson correlation analysis: the correlation was strong (r = 0.751; P < 0.0001).
We conclude that use of an antigen retrieval methodology on paraffin-embedded breast cancer samples gave similar results to those observed with frozen breast tissues. Because hENT1 deficiency has been associated with decreased cellular sensitivity to nucleoside chemotherapy (2), this new hENT1 immunohistochemical assay will allow us to determine the relationship of the hENT1 protein abundance in routinely available clinical specimens. This will, in turn, allow us to determine whether clinical outcomes after nucleoside chemotherapy are related to pretreatment hENT1 protein abundance assessed by immunohistochemistry.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviation used is: hENT1, human equilibrative nucleoside transporter 1.