The Estrogen Receptor Negative-Progesterone Receptor Positive Breast Carcinoma is a Biological Entity and not a Technical Artifact

Steroid hormone receptors (HR) are important prognostic and predictive markers for response to endocrine therapy in the management of breast cancer. Several studies have found that up to 10% of estrogen receptor-negative (ER-) breast cancers are progesterone receptor-positive (PR+) (Rakha et al., 2010) although recent evidence shows that the percentage is much lower when more sensitive immunohistochemical (IHC) methods for ER determination are used (Rakha et al., 2007; 2010; Rhodes & Jasani 2009) and the high proportion of ER-/PR+ may be due to a false-negative ER assay (Nadji et al., 2005; Nadji, 2008). There have been differing opinions on the status of ERin the presence of PR+ status in breast tumors over the last few years. The ER-/PR+ phenotype is simply thought by some to be due to artifacts arising from the preparation or assay of the sample i.e. due to inadequate tissue fixation or technique failure of the IHC assay, and that these tumors are essentially positive for both receptors (Nadji et al., 2005; Nadji, 2008). If this is true, then it would be expected that the ER-/PR+ phenotype would have the same tumor characteristics as the ER+/PR+ phenotype. However, recentstudies have


Introduction
Steroid hormone receptors (HR) are important prognostic and predictive markers for response to endocrine therapy in the management of breast cancer.Several studies have found that up to 10% of estrogen receptor-negative (ER-) breast cancers are progesterone receptor-positive (PR+) (Rakha et al., 2010) although recent evidence shows that the percentage is much lower when more sensitive immunohistochemical (IHC) methods for ER determination are used (Rakha et al., 2007;2010;Rhodes & Jasani 2009) and the high proportion of ER-/PR+ may be due to a false-negative ER assay (Nadji et al., 2005;Nadji, 2008).There have been differing opinions on the status of ER-in the presence of PR+ status in breast tumors over the last few years.The ER-/PR+ phenotype is simply thought by some to be due to artifacts arising from the preparation or assay of the sample i.e. due to inadequate tissue fixation or technique failure of the IHC assay, and that these tumors are essentially positive for both receptors (Nadji et al., 2005;Nadji, 2008).If this is true, then it would be expected that the ER-/PR+ phenotype would have the same tumor characteristics as the ER+/PR+ phenotype.However, recent-studies have shown that ER-/PR+ tumors exhibit more aggressive behavior than double-hormone receptor-positive cancers (Rakha et al., 2007).The aim of this study was therefore to investigate whether there is a difference in patient and tumor characteristics between breast cancers of differing steroid hormone receptor status in an Asian setting.

Materials and Methods
The University Malaya Medical Centre (UMMC)'s prospective Breast Cancer Registry was used to identify 2629 patients newly diagnosed from 2003-2009 with known ER and PR status.Patients were divided into four categories namely ER+/PR+, ER+/PR+, ER-/PR+ and ER-/PR-.Factors studied in relation to these four groups were age, ethnicity (Chinese, Malay, Indian), tumor grade (Bloom-Richardson; grade 1, 2, 3) and histological type (invasive ductal, invasive lobular, others).
The estrogen and progesterone receptor status of each case was determined immunohistochemically utilizing the antibody clones 1D5 (Dako Ltd, Denmark) and PgR 636 (Dako Ltd Denmark), respectively.Briefly ,the slides were de-paraffinised and endogenous peroxidase blocked in 0.3% hydrogen peroxide prior to microwave antigen retrieval in 0.01M sodium citrate (pH 6.0) buffer for a duration of 30 minutes.Following incubation in primary antibody clones for 60 minutes, detection was achieved by placing in an EnVision polymer with HRP label (Dako, Ltd, Denmark) solution for a further30 min.Visualisation was achieved by using a hydrogen peroxide substrate a diaminobenzidine chromogen (Dako Ltd, Denmark).Nuclei were counterstained with Harris's Haematoxylin.
Categorical variables were compared using the chi-square test -and continuous variables using one way analysis of variance (ANOVA).Univariable and multivariable logistic regression analyses were used to determine the association between patient and tumor characteristics (independent) and hormone receptor status (dependent).All analyses were performed using SPSS version 16.0 (SPSS Inc, Chicago, USA).
In the univariable logistic regression, patients with ER-/PR+ tumors were 2.5 times more likely to be younger than 50 years at diagnosis compared to all other phenotypes; OR: 2.52; 95% CI: 1.72 -3.67.
The significance of the single HR+ phenotype that includes ER+/PR-and ER-/PR+ is still poorly understood.The percentage of ER+/PR-(11.6%) and ER-/PR+ (4.6%) tumors in our cohort is found to be consistent to those quoted in other studies.These tumors are often of higher grade, larger size and hormonal therapies are less likely to be effective than in the ER+/PR+ phenotype (Rakha, et al., 2007;De et al., 2008;Rakha et al., 2010).In this study we found that the ER-/PR+ tumor was significantly associated with a younger age at diagnosis compared to all other phenotypes.A similar finding was found (Rakha et al., 2007) who showed that the majority of the ER+/ PR-phenotypes were significantly older as compared to the ER-/PR+ phenotype.Our result is also consistent with previous studies that showed the ER-/PR+ phenotype occurring twice as often in the less than 50-year age group as compared to those 50 years of age and above (De Maeyer et al. 2008;Rhodes and Jasani 2009).If the ER-/ PR+ phenotype does not exist and is indeed an artifact of the ER or PR assay then there should be no difference in the age at diagnosis.
Studies have shown that single hormone receptor positive phenotype which include ER+/PR-and ER-/ PR+ were more often of higher histology grade and larger in size, more likely to be aneuploid and show higher expression of proliferation-related genes than ER+/ PR+ and both single HR+ groups are similar and they both might have biological characteristics somewhere in between ER+/PR+ and ER-/PR- (De et al., 2008;Rakha et al., 2010).We found that higher grades of tumors are more likely to be associated with ER-/PR+ as compared with ER+/PR+ tumors but less likely to be associated with ER-/ PR-tumors in this series and more likely to have invasive lobular than invasive ductal histology.This is the first time that the ER-/PR+ has been shown to be associated with invasive lobular carcinoma (ILC).However this finding has to be interpreted with caution because ILC comprised only 8.9% of the breast cancers in this study.It is known that infiltrating lobular carcinomas are less common in Asians compared to Caucasians (Yip 2009).
In conclusions, the ER-/PR+ phenotype is significantly associated with a younger age at diagnosis compared to all other phenotypes.It also is associated with intermediate histopathological characteristics compared to ER+/PR+ and ER-/PR-tumors, whereby it is more aggressive than ER+/PR+ tumor but less aggressive than ER-/PR-tumors and therefore unlikely to be a technical artifact.