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The Estrogen Receptors , ß, and ßcx

Academic Unit of Pathology, University of Leeds, Leeds, United Kingdom

Valerie Speirs

Molecular Medicine Unit, University of Leeds, Leeds, United Kingdom

To the Editor: We read with interest the article by Esslimani-Sahla et al. (1) who report a progressive decrease of estrogen receptor (ER) ß1 expression from normal breast to ductal carcinoma in situ (DCIS) with further increase from DCIS to invasive carcinoma. In contrast, expression of ERßcx increased during carcinogenesis. In our series, composed of 53 normal breasts, 54 hyperplasia of usual type, 35 DCIS examples, and 141 invasive carcinomas, and using an antibody specific for ERß1, we showed significant decrease in ERß1 expression from normal breast through hyperplasia of usual type to DCIS. However, there was no significant difference between DCIS and invasive carcinoma in this study (2) and in an independent study by our group, composed of 138 normal breasts, 16 pure DCIS, 319 invasive cancers, and 39 recurrences, and using a pan-specific ERß antibody for detection of ERß1 and variants (3). There is no comment on the variation of ERß1 and ERßcx expression in different grades of DCIS. In a few studies examining ERß1 expression in various grades of DCIS, a decrease in ER and ERß1 expression was observed in high-grade DCIS (n = 59; ref. 4) whereas we showed no significant difference in ERß expression between low-grade and high-grade DCIS (n = 35; ref. 3).

One of the concerns about the emergence of ERß is the lack of a standardized scoring system for its immunohistochemical detection, with many being reported (5). Here, the authors used a quantitative score to express the immunohistochemistry results. How was this score formalized? It would have been useful to use a cutoff value for expression to allow comparison with previous studies. Furthermore, we have consistently reported cytoplasmic staining, in addition to nuclear expression, as a feature of ERß1 immunohistochemistry (2, 6) and this is likely to reflect genuine expression rather than artifact. The authors do not state whether this phenomenon also applies to ERßcx. On a technical note, it is remarkable that in the protocol of immunostaining, the duration of pressure cooking for antigen retrieval was 15 minutes. The average duration of pressure cooking in most pathology laboratories is 2.5 minutes. We have used this successfully with the same buffer described by the authors for our previous immunohistochemical studies of ERß1 (2, 6). Extensive antigen retrieval carries a risk of damage to the protein of interest, which might result in nonspecific staining and spurious high levels of expression.

Whereas the lack of a standardized scoring method for ERß is recognized, overall, the Allred method is the most widely accepted although with variation in the cutoff used to define positivity (7). Development of a uniform scoring system should be a priority to allow us to determine if ERß might have therapeutic implications in breast cancer.

References

1. Esslimani-Sahla M, Kramar A, Simony-Lafontaine J, Warner M, Gustafsson JA, Rochefort H. Increased estrogen receptor ßcx expression during mammary carcinogenesis. Clin Cancer Res 2005;11:3170–4.[Abstract/Free Full Text]
2. Shaaban AM, O'Neill PA, Davies MP, et al. Declining estrogen receptor-ß expression defines malignant progression of human breast neoplasia. Am J Surg Pathol 2003;27:1502–12.[Medline]
3. Skliris GP, Munot K, Bell SM, et al. Reduced expression of oestrogen receptor ß in invasive breast cancer and its re-expression using DNA methyltransferase inhibitors in a cell line model. J Pathol 2003;201:213–20.[CrossRef][Medline]
4. Roger P, Sahla ME, Makela S, Gustafsson JA, Baldet P, Rochfort H. Decreased expression of estrogen receptor ß protein in proliferative preinvasive mammary tumors. Cancer Res 2001;61:2537–41.[Abstract/Free Full Text]
5. Speirs V, Carder PJ, Lane S, Dodwell D, Lansdown MRJ, Hanby AM. Oestrogen receptor ß: what it means for patients with breast cancer. Lancet Oncol 2004;5:174–81. Erratum in: Lancet Oncol 2004;5:208.[CrossRef][Medline]
6. O'Neill PA, Davies MP, Shaaban AM, et al. Wild-type oestrogen receptor ß (ERß1) mRNA and protein expression in Tamoxifen-treated post-menopausal breast cancers. Br J Cancer 2004;91:1694–702.[Medline]
7. Carder PJ, Murphy CE, Dervan P, et al. A multi-centre investigation towards reaching a consensus on the immunohistochemical detection of ERß in archival formalin-fixed paraffin embedded human breast tissue. Breast Cancer Res Treat. 2005;92:287–93.[Medline]

Institu National de la Sante, et de la Recherche Medicale U540, Montpellier, France

In Response: A.M. Shabaan and V. Speirs raised several questions about our article (1):

1. The reason for the discrepancy with the Skliris et al. study (2) concerning the difference in total ERß expression in ductal carcinoma in situ (DCIS) and invasive carcinoma was already mentioned in our discussion (1) and is not the major focus of our study. We previously found much lower median values for total ERß expression in DCIS (60 cases; ref. 3) than in invasive carcinoma (50 cases; ref. 4). This increased expression of total ERß from DCIS to invasive carcinoma was also shown comparatively in 43 cases in ref. (1). This might be due to an increased expression of variants (ERßcx ?). This discrepancy should be solved by other independent studies on a larger scale population and using ERß1-specific antibodies.
   
2. We could not comment on a possible difference between ERßcx expression in low and high nuclear grade DCIS, the number of cases being too small in our study. However, we previously found (3) a significantly lower expression of total ERß in high-grade compared to low-grade DCIS.
   
3. The validity of our ERßcx assay has been shown previously (4) and is confirmed by several criteria in ref. (1). In our experiments, the cytoplasmic ERßcx staining was not totally abolished by adding an excess of antigen or by using irrelevant antibodies, contrary to the nuclear staining. We have therefore only quantified the nuclear staining. Following the recommendations made by Shi et al. (5), 15 minutes of pressure cooking antigen retrieval was chosen as our optimal time conditions for specific ERßcx expression without tissue detachment from the slides and without loss of specificity. We agree that this time might not be the best for other conditions (5).
   
4. We used a computer-aided image analyzer to quantify ERß and ERßcx levels in a continuous scale. The method has been described in several of our previous reports (3, 6) and presents several advantages for research studies:

   (a) Results are given in a continuous scale. This avoids the need to choose an arbitrary cutoff level.
   

   (b) It is more objective, with the pathologist first determining the region of tissue to be quantified (i.e., the normal, premalignant, and cancer epithelial cells), the level of staining then being calculated more objectively on a large number of cells. Results are given as a percentage of positive nuclei and in QIC score integrating the percentage of positive nuclei and the staining intensity as described in refs. (1, 6). This quantitative immunohistochemical method has been proven to be superior to visual estimates, and may become a tool for more accurate interlaboratory and intralaboratory quality control, as mentioned in ref. (7). However, a major drawback is that this quantification is time-consuming and may not be adapted for routine analysis. We therefore fully agree with Shabaan and Speirs that we need a standardized scoring system for immunohistochemical detection of ERß and variants for routine analysis in invasive breast cancer.
   

   
   

In conclusion, the major and new information of our study is the increased level of ERßcx variant contrasting with the decreased level of total ERß from normal to cancer cells (most laboratories now agree on this last point). This raises several interesting questions on the mechanism and consequence of the ERß differential splicing in human breast cancer. On this point, we have not seen any discrepancy with Shabaan and Speirs' comments.

References

1. Esslimani-Sahla M, Kramar A, Simony-Lafontaine J, Warner M, Gustafsson J-A, Rochefort H. Increased estrogen receptor ßcx expression during mammary carcinogenesis. Clin Cancer Res 2005;11:3170–4.
2. Skliris GP, Munot K, Bell SM, et al. Reduced expression of estrogen receptor ß in invasive breast cancer and its re-expression using DNA methyl transferase inhibitors in a cell line model. J Pathol 2003;201:213–20.
3. Roger P, Esslimani Sahla M, Makela S, Gustafsson J-Å, Baldet P, Rochefort H. Decreased expression of estrogen receptor ß protein in proliferative preinvasive mammary tumors. Cancer Res 2001;61:2537–41.
4. Esslimani-Sahla M, Simony-Lafontaine J, Kramar A, et al. ERß level helps to predict Tam resistance in breast cancer but not its ERßcx variant. Clin Cancer Res 2004;10:5769–76.[Abstract/Free Full Text]
5. Shi S-R, Cote RJ, Taylor CR. Antigen retrieval immunohistochemistry: past, present and future. J Histochem Cytochem 1997;45:3527–43.
6. Maudelonde T, Brouillet JP, Roger P, Giraudier V, Pages A, Rochefort H. Immunostaining of cathepsin D in breast cancer: quantification by computerized image analysis and correlation with cytosolic assay. Eur J Cancer 1992;28A:1686–91.[CrossRef]
7. Seidal T, Balaton AJ, Battifora H. Interpretation and quantification of immunostains. Am J Surg Pathol 2001;25:1204–7.[CrossRef][Medline]

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