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In Reply

Molecular Medical Sciences, University of Nottingham, Department of Histopathology, Nottingham City Hospital NHS Trust, Nottingham, United Kingdom

In the letter from De Maeyer et al, the authors indicate that the estrogen receptor (ER)–negative/progesterone receptor (PR)–positive group does not exist and appears to be an artifact of the use of arbitrary thresholds for positivity and technical method sensitivity. To address the points raised by De Maeyer et al, we need to consider the definition of ER positivity and negativity in research and routine practice, given the value of determination of ER expression on prediction of response to hormone therapy and patients' outcome. Clearly, this is an important issue for pathologists, medical oncologists, and patients.

ER has a documented technical false-negative rate,^1,2 which may be increased by the use of tissue microarrays in research studies, the approach we have adopted in our study.³ Although we accept the limitations of tissue microarrays for immunohistochemical (IHC) detection of ER, their use is the only practical method for screening of biomarker expression in large case series. This approach is supported by several lines of evidence that the agreement of ER status between whole tissue sections and tissue microarrays is more than 90% using a single core.⁴ In the breast cancer literature, a wide variety of scoring systems for ER and PR have been used that include both of qualitative⁵ and quantitative⁶ estimations, and no uniformly accepted cutoff point for positivity has been determined for IHC evaluation.^7,8 In addition, previous studies, which addressed the concordance of different methods of ER assessment, have reported discordance between ligand-binding assay and IHC, between different ER antibodies used for IHC,⁹ and between locally versus centrally assessed hormone receptors (HR) status.⁸

De Maeyer et al stained 32 breast cancer cases, which were previously diagnosed in their routine practice as ER-negative/PR-positive, using a more sensitive IHC technique and recorded any positive nuclear reaction for ER and PR, irrespective of percentage of reactive cells, as positive. This cutoff of ER-positivity (> 0%) has also been used by Nadji et al,² who also reported that ER-negative/PR-positive tumors do not exist. It is also supported by some studies indicating a bimodal distribution of HR status. However, there are several lines of evidence to demonstrate a direct, almost linear relationship between the amount of ER protein present in tumor cells and the amount of ER antigen detected by IHC. Furthermore, quantification of ER by IHC may be of value in helping to identify patients with ER-positive tumors who may benefit most from adjuvant endocrine therapy and potentially assist in assessment of use of chemotherapy.¹⁰ As emphasized by Schnitt et al, who pointed out that when highly sensitive, possibly oversensitive, anti-ER antibodies and detection systems are used on formalin-fixed tissue samples, IHC methods may lose their semiquantitative power. Ellis et al¹¹ have reported a linear relationship between the level of ER expression and response to both tamoxifen and letrozole in the neoadjuvant setting. Previous studies have demonstrated that the response to hormone therapy is associated with the levels of both ER and PR.^10,12,13 Given that the response to endocrine therapy is associated with levels of both ER and PR, a comparison of absolute HR levels (as opposed to a dichotomous classification; positive v negative) might help to clarify why some patients do not respond to hormone therapy. In addition, Harvey et al,¹⁴ who provided an excellent overview of the IHC problem, has espoused the need to base cutoff of ER positivity on patient outcome.

In our series, the ER-negative/PR-positive group constituted 3.4%, which is similar to that reported by several previous studies, which showed ER-negative/PR-positive class constitutes 2% to 7% of breast cancer.¹⁵-²¹ Other reports¹⁷-²⁰, ²², ²³ like ours³ have determined that ER-negative/PR-positive is a distinct class of breast cancer, which is associated with clinicopathological variables and patient outcome worse than both ER-positive/PR-positive and ER-positive/PR-negative, but better than ER-negative/PR-negative tumors. These results provide evidence that this class is a distinct biologic and clinical entity that is different from ER-positive tumors as a whole. In our study (Table 4A),³ the median level of PR expression in the ER-negative/PR-positive tumors was 85% (range, 7% to 100%); in 48 cases (80%), PR expression was higher than 20% (Table 4A),³ indicating that the PR positivity at least in the majority of ER-negative/PR-positive tumors is moderate to strong, and therefore, the possibility of false PR positivity appears limited.

To address directly the question raised by De Maeyer et al, and to assess the existence of ER-negative/PR-positive tumors in our series, we have stained large whole tissue sections from all ER-negative/PR-positive cancers (60 cases) in addition to an equal number of randomly selected tumors from ER-negative/PR-negative group using the same sensitive staining method and scoring system used by De Maeyer et al. Staining of ER-negative/PR-positive tumors confirmed the original results (ER-negative/PR-positive) in 27 cases. False negative staining of ER was found in 12 cases (changed to ER-positive/PR-positive) and false positive (due to interpretive errors) PR in four cases (changed to ER-negative/PR-negative). In 17 cases, there was focal weak staining of ER while that of PR was typically strong and mainly diffuse. Interestingly, in the double negative control group, seven cases also showed positive ER staining, usually focal and weak, and two cases showed focal positivity for PR. Therefore, according to the new staining results based on conventional large section examination, 29 cases were confirmed to be ER-negative/PR-positive (1.6%). Importantly, when we repeated our analysis after changing the classification of ER/PR cases according to these results, similar associations with clinicopathological variables and patients' outcome to that in our article³ were obtained. We therefore agree with the general observation in breast cancer that improvements in sensitivity of IHC methodology have reduced the numbers of cases deemed ER-negative. We have, however, in this large series of cases, confirmed that even using the most sensitive methods examples of ER-negative/PR-positive breast cancer exist, but are rare, and that they have distinct clinicopathological characteristics

Biologically, we speculate that PR could be regulated by a mechanism independent of ER-negative– in these ER-negative/PR-positive cells, possibly by some aberrant growth factor signaling pathway, and where the underlying signaling also permits aggressive tumor behavior; taking into account the different ER and PR isoforms and the possible regulation of PR promoter activity by cAMP and transcription factors such as SP-1 and AP-1. Previously, ER-negative/PR-positive breast cancer cell lines have been described.²⁴ In our previous experiment on FASRLT sublines that were generated by prolonged fulvestrant exposure (unpublished data), we found that these cells lines acquired resistance and underwent epithelial mesenchymal transition/gain of aggressive features and complete ER loss at the protein and mRNA level. These cells, which have a highly aggressive phenotype and were initially ER-negative/PR-negative, appeared to acquire PR staining in further long-term culture despite not gaining ER. These observations are therefore, consistent with our clinical findings, which indicate that not only the ER-negative/PR-negative phenotype, but also the ER-negative/PR-positive phenotype can be aggressive and associate with endocrine resistance.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The author(s) indicated no potential conflicts of interest.

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