J. Comp. Path. 2014, Vol. -, 1e9
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Evaluation of Clinicopathological Characteristics and Oestrogen Receptor Gene Expression in Oestrogen Receptor-negative, Progesterone Receptor-positive Canine Mammary Carcinomas N.-H. Kim*,†, H.-Y. Lim*, K.-S. Im*, J.-I. Shin*, H.-W. Kim* and J.-H. Sur* * Department of Veterinary Pathology, Small Animal Tumour Diagnostic Centre, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701 and † Gyeongnam Department of Environment and Toxicology, Korea Institute of Toxicology, 17 Jegok-gil, Munsan-eup, Gyeongnam 660-844, Republic of Korea
Summary The existence of the oestrogen receptor-negative (OR)/progesterone receptor-positive (PR+) phenotype in canine mammary carcinomas (CMCs) is not well understood, although this phenotype was reported consistently in previous studies. In the present study, immunohistochemistry (IHC) was performed to categorize CMCs with the OR/PR+ phenotype and compare their clinicopathological features with OR+/PR+ tumours. Of a total of 305 CMCs, 36 (11.8%) were categorized as OR/PR+ and showed intermediate characteristics between those of OR+/PR+ and OR/PR cases. OR mRNA levels were measured in formalin-fixed, paraffin wax-embedded samples using a novel branched-chain DNA assay method. Similar to the IHC result, one-way analysis of variance showed that the mean normalized OR mRNA level of OR/PR+ tumours (11.4 16.34) was between that of the OR/PR (mean 4.7 6.35) and OR+/PR+ (mean 15.8 11.95) (P ¼ 0.033) tumours. Only three of the 36 OR/PR+ tumours completely lacked OR mRNA expression. The OR/PR+ tumours were not categorized as an independent group nor were they included in the other groups on post-hoc analysis. OR/PR+ tumours were associated with factors related to poor prognosis compared with OR+/PR+ tumours, but OR/PR tumours were associated with the worst prognostic indicators. Further studies are required in order to determine the clinical significance of the OR/PR+ phenotype. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: branched-chain DNA assay; canine mammary carcinoma; steroid receptors
Introduction Canine mammary gland tumours are the most common neoplasms in female dogs (Misdorp et al., 1999), but these tumours have heterogeneous features (Rutteman et al., 2001; Misdorp, 2002). Clinical and pathological factors including histological type and grade, lymph node status, distant metastasis, tumour size and age are known to affect the prognosis for canine mammary gland tumours (Yamagami et al., 1996; Rutteman et al., 2001; *Correspondence to: J.-H. Sur (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.04.001
Misdorp, 2002; Sarli et al., 2002). Research on the molecular mechanisms underlying tumour development has identified other factors that may be associated with tumour growth and progression, although much remains unclear (Pena et al., 1998; Wakui et al., 2001; Sarli et al., 2002). The steroid receptors, oestrogen receptor (OR) and progesterone receptor (PR) are members of the nuclear receptor superfamily and play important roles as transcription factors and in signal transduction of steroid hormones. OR and PR are considered potent prognostic factors for human breast cancer (Murphy and Watson, 2002) and canine mammary Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kim N-H, et al., Evaluation of Clinicopathological Characteristics and Oestrogen Receptor Gene Expression in Oestrogen Receptor-negative, Progesterone..., Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.04.001
2
N.-H. Kim et al.
carcinomas (CMCs) (Sartin et al., 1992; Nieto et al., 2000; Chang et al., 2009). In particular, OR expression is known to be closely related to mammary tumourigenesis, while PR expression is primarily an indicator of intact OR function (Murphy and Watson, 2002). In human breast cancer, the expression of steroid receptors has prognostic value and predictive value for the response to endocrine therapy (Pichon et al., 1996; Yamashita et al., 2006). Breast cancers with low or negative expression of OR and PR are not sensitive to endocrine therapy and are associated with a poor clinical outcome (Osborne et al., 2005), while steroid receptor-positive tumours have a relatively favourable prognosis (Lester, 2004; Dowsett et al., 2006). In CMCs, a number of studies of steroid receptor expression have found similar results to those reported for human breast cancers (Sartin et al., 1992; Nieto et al., 2000); however, few of these reports have focused on the combined expression of both OR and PR. Martin de Las Mulas et al. (2005) showed that tumours with expression of both ORa and PR were associated with a longer disease-free period than tumours that lacked expression of both receptors, and Chang et al. (2009) showed that tumours with the OR+/PR+ phenotype had the highest survival rates when compared with other phenotypes. Breast cancers that express both OR and PR have been shown to have favourable outcomes, but the characteristics of tumours that express only one of these steroid receptors are not well defined (Rakha et al., 2007). Tumours with an OR+/PR phenotype are believed to be a distinct subset of tumours that are resistant to endocrine therapy despite OR positivity (Thakkar and Mehta, 2011). Since the loss of PR expression implies that normal OR function is impaired, there is decreased response to the OR antagonist tamoxifen (Belleine et al., 2000; Cui et al., 2005). The existence of OR/PR+ tumours is controversial because PR is typically known to be expressed under OR+ conditions (De Maeyer et al., 2008; Cserni et al., 2011) and the specific characteristics of OR/PR+ CMCs have not been discussed in the veterinary field. The aim of the present study was to categorize CMCs by their OR/PR phenotype using immunohistochemistry (IHC) and to evaluate the clinicopathological features of the different tumour phenotypes. A further aim was to determine whether the OR/PR+ phenotype is truly OR by measuring OR gene expression with a branched-chain DNA assay and comparing these results with those from OR+/PR+ and OR/PR tumours.
Materials and Methods Tissue Samples
In total, 305 samples from cases of primary CMC were categorized for OR and PR expression by IHC. Mesenchymal tumours of the mammary gland (e.g. fibrosarcoma and osteosarcoma) were excluded. All samples were obtained from the histopathological database of the Department of Veterinary Pathology, Veterinary Medical Teaching Hospital of Konkuk University, Seoul, Korea, between 2010 and 2012. Immunohistochemistry
Sections (4 mm) were cut from formalin-fixed and paraffin wax-embedded (FFPE) tissue samples, dewaxed in xylene, rehydrated in graded ethanols and then washed in phosphate buffered saline (PBS, pH 7.4; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). Endogenous peroxidase activity was blocked in H2O2 3% in PBS for 20 min at room temperature. Antigen retrieval was performed by boiling for 15 min in triseEDTA (pH 9.0) for OR or citric acid (pH 6.0) for PR in a microwave oven. After washing three times in PBS, primary antibodies were applied to the slides. Anti-OR antibodies (Clone ER88, Biogenex, San Ramon, California, USA) were diluted 1 in 60 and incubated for 3 h at room temperature. Anti-PR antibodies (Clone 10A9, Immunotech SAS, Marseille, France) were diluted 1 in 500 and incubated at 4 C overnight. For the anti-OR antibodies only, 5% normal goat serum was used as a blocking agent. All slides were washed four times in PBS, and were then incubated with horseradish peroxidase-conjugated secondary antibodies (DAKO REALÔ Envision kit; DAKO, Glostrup, Denmark) for 40 min. Mouse IgG1 (eBioscience, San Diego, California, USA) and mouse IgG2a (Biolegend, San Diego, California, USA) were used as isotype controls. A tumour was considered positively labelled when OR or PR were expressed by >10% of the nuclei in the entire section of the tumour. Tumour Classification
The CMCs were evaluated based on the classification proposed by Goldschmidt et al. (2011) and were subcategorized into three groups: epithelial tumours, special types and mixed tumours. Three histological grades according to Elston and Ellis (1991) were used: grade I, well differentiated; grade II, moderately differentiated; and grade III, poorly differentiated. The cases were also categorized by
Please cite this article in press as: Kim N-H, et al., Evaluation of Clinicopathological Characteristics and Oestrogen Receptor Gene Expression in Oestrogen Receptor-negative, Progesterone..., Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.04.001
Oestrogen Receptor Gene Expression in Canine Mammary Carcinoma
clinicopathological parameters including the presence of central necrosis, lymphatic vessel infiltration, age of the animal and tumour size. Oestrogen Receptor Gene Expression
Levels of OR mRNA were compared in 36 OR/ PR+ tumours, 20 OR/PR tumours and 20 OR+/PR+ tumours. The QuantiGene 2.0 Reagent System was used according to the manufacturer’s protocol. For tissue homogenization, approximately 100e200 mm2 of tumour area, 50 mm thick, was selectively dissected per FFPE sample. Tissue sections were incubated for 5 h with 300 ml of homogenizing solution and 4.5 ml proteinase K in a thermomixer at 65 C and 850 revolutions per minute (Eppendorf, Hamburg, Germany). The homogenates were separated from wax and tissue debris by centrifugation, transferred to a new tube and stored at 70 C until used. Probe set designs for the target gene (canine OR 1; XM_533454) and the reference gene (canine GAPDH; NM_001003142) were requested from Panomics Inc., Fremont, California, USA. To capture the target RNA, the working probe set was prepared in proportion to the number of wells to be run by mixing 33.3 ml of lysis mixture, 25.4 ml nuclease-free water, 1 ml of blocking reagent and 0.3 ml of probe set per well. Forty microlitres of tissue homogenate and 60 ml of working probe set were dispensed into each oligonucleotide-conjugated well of the 96-well capture plate. The plate was incubated for 16 h at 55 C, followed by washing of each well three times with wash buffer. For signal amplification, 100 ml each of 2.0 preamplifier working reagent, 2.0 amplifier working reagent and label probe working reagent were applied consecutively to each well and hybridized for 1 h at 55 C, 55 C and 50 C, respectively. Wells were washed three times with wash buffer between each step. For signal detection, 100 ml of 2.0 substrate was applied to each well and the plate was sealed and incubated at room temperature for 5 min. The luminescence of each well was measured by a luminometer (Turner Biosystems, Sunnyvale, California, USA). To determine assay linearity, each sample was run as a twofold dilution. To normalize the data, the following formula was used: normalized OR ¼ 1,000 (average value for OR average value for background control)/value for canine GAPDH. Statistical Analysis
Pearson’s chi-square test and one-way analysis of variance (ANOVA) with post-hoc test were per-
3
formed to identify the relevance of combined expression of the hormone receptors and clinicopathological parameters: histological type, grade, central necrosis, lymphatic infiltration, age and tumour size. P
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Evaluation of Clinicopathological Characteristics and Oestrogen Receptor Gene Expression in Oestrogen Receptor-negative, Progesterone Receptor-positive Canine Mammary Carcinomas N.-H. Kim*,†, H.-Y. Lim*, K.-S. Im*, J.-I. Shin*, H.-W. Kim* and J.-H. Sur* * Department of Veterinary Pathology, Small Animal Tumour Diagnostic Centre, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701 and † Gyeongnam Department of Environment and Toxicology, Korea Institute of Toxicology, 17 Jegok-gil, Munsan-eup, Gyeongnam 660-844, Republic of Korea
Summary The existence of the oestrogen receptor-negative (OR)/progesterone receptor-positive (PR+) phenotype in canine mammary carcinomas (CMCs) is not well understood, although this phenotype was reported consistently in previous studies. In the present study, immunohistochemistry (IHC) was performed to categorize CMCs with the OR/PR+ phenotype and compare their clinicopathological features with OR+/PR+ tumours. Of a total of 305 CMCs, 36 (11.8%) were categorized as OR/PR+ and showed intermediate characteristics between those of OR+/PR+ and OR/PR cases. OR mRNA levels were measured in formalin-fixed, paraffin wax-embedded samples using a novel branched-chain DNA assay method. Similar to the IHC result, one-way analysis of variance showed that the mean normalized OR mRNA level of OR/PR+ tumours (11.4 16.34) was between that of the OR/PR (mean 4.7 6.35) and OR+/PR+ (mean 15.8 11.95) (P ¼ 0.033) tumours. Only three of the 36 OR/PR+ tumours completely lacked OR mRNA expression. The OR/PR+ tumours were not categorized as an independent group nor were they included in the other groups on post-hoc analysis. OR/PR+ tumours were associated with factors related to poor prognosis compared with OR+/PR+ tumours, but OR/PR tumours were associated with the worst prognostic indicators. Further studies are required in order to determine the clinical significance of the OR/PR+ phenotype. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: branched-chain DNA assay; canine mammary carcinoma; steroid receptors
Introduction Canine mammary gland tumours are the most common neoplasms in female dogs (Misdorp et al., 1999), but these tumours have heterogeneous features (Rutteman et al., 2001; Misdorp, 2002). Clinical and pathological factors including histological type and grade, lymph node status, distant metastasis, tumour size and age are known to affect the prognosis for canine mammary gland tumours (Yamagami et al., 1996; Rutteman et al., 2001; *Correspondence to: J.-H. Sur (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.04.001
Misdorp, 2002; Sarli et al., 2002). Research on the molecular mechanisms underlying tumour development has identified other factors that may be associated with tumour growth and progression, although much remains unclear (Pena et al., 1998; Wakui et al., 2001; Sarli et al., 2002). The steroid receptors, oestrogen receptor (OR) and progesterone receptor (PR) are members of the nuclear receptor superfamily and play important roles as transcription factors and in signal transduction of steroid hormones. OR and PR are considered potent prognostic factors for human breast cancer (Murphy and Watson, 2002) and canine mammary Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kim N-H, et al., Evaluation of Clinicopathological Characteristics and Oestrogen Receptor Gene Expression in Oestrogen Receptor-negative, Progesterone..., Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.04.001
2
N.-H. Kim et al.
carcinomas (CMCs) (Sartin et al., 1992; Nieto et al., 2000; Chang et al., 2009). In particular, OR expression is known to be closely related to mammary tumourigenesis, while PR expression is primarily an indicator of intact OR function (Murphy and Watson, 2002). In human breast cancer, the expression of steroid receptors has prognostic value and predictive value for the response to endocrine therapy (Pichon et al., 1996; Yamashita et al., 2006). Breast cancers with low or negative expression of OR and PR are not sensitive to endocrine therapy and are associated with a poor clinical outcome (Osborne et al., 2005), while steroid receptor-positive tumours have a relatively favourable prognosis (Lester, 2004; Dowsett et al., 2006). In CMCs, a number of studies of steroid receptor expression have found similar results to those reported for human breast cancers (Sartin et al., 1992; Nieto et al., 2000); however, few of these reports have focused on the combined expression of both OR and PR. Martin de Las Mulas et al. (2005) showed that tumours with expression of both ORa and PR were associated with a longer disease-free period than tumours that lacked expression of both receptors, and Chang et al. (2009) showed that tumours with the OR+/PR+ phenotype had the highest survival rates when compared with other phenotypes. Breast cancers that express both OR and PR have been shown to have favourable outcomes, but the characteristics of tumours that express only one of these steroid receptors are not well defined (Rakha et al., 2007). Tumours with an OR+/PR phenotype are believed to be a distinct subset of tumours that are resistant to endocrine therapy despite OR positivity (Thakkar and Mehta, 2011). Since the loss of PR expression implies that normal OR function is impaired, there is decreased response to the OR antagonist tamoxifen (Belleine et al., 2000; Cui et al., 2005). The existence of OR/PR+ tumours is controversial because PR is typically known to be expressed under OR+ conditions (De Maeyer et al., 2008; Cserni et al., 2011) and the specific characteristics of OR/PR+ CMCs have not been discussed in the veterinary field. The aim of the present study was to categorize CMCs by their OR/PR phenotype using immunohistochemistry (IHC) and to evaluate the clinicopathological features of the different tumour phenotypes. A further aim was to determine whether the OR/PR+ phenotype is truly OR by measuring OR gene expression with a branched-chain DNA assay and comparing these results with those from OR+/PR+ and OR/PR tumours.
Materials and Methods Tissue Samples
In total, 305 samples from cases of primary CMC were categorized for OR and PR expression by IHC. Mesenchymal tumours of the mammary gland (e.g. fibrosarcoma and osteosarcoma) were excluded. All samples were obtained from the histopathological database of the Department of Veterinary Pathology, Veterinary Medical Teaching Hospital of Konkuk University, Seoul, Korea, between 2010 and 2012. Immunohistochemistry
Sections (4 mm) were cut from formalin-fixed and paraffin wax-embedded (FFPE) tissue samples, dewaxed in xylene, rehydrated in graded ethanols and then washed in phosphate buffered saline (PBS, pH 7.4; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). Endogenous peroxidase activity was blocked in H2O2 3% in PBS for 20 min at room temperature. Antigen retrieval was performed by boiling for 15 min in triseEDTA (pH 9.0) for OR or citric acid (pH 6.0) for PR in a microwave oven. After washing three times in PBS, primary antibodies were applied to the slides. Anti-OR antibodies (Clone ER88, Biogenex, San Ramon, California, USA) were diluted 1 in 60 and incubated for 3 h at room temperature. Anti-PR antibodies (Clone 10A9, Immunotech SAS, Marseille, France) were diluted 1 in 500 and incubated at 4 C overnight. For the anti-OR antibodies only, 5% normal goat serum was used as a blocking agent. All slides were washed four times in PBS, and were then incubated with horseradish peroxidase-conjugated secondary antibodies (DAKO REALÔ Envision kit; DAKO, Glostrup, Denmark) for 40 min. Mouse IgG1 (eBioscience, San Diego, California, USA) and mouse IgG2a (Biolegend, San Diego, California, USA) were used as isotype controls. A tumour was considered positively labelled when OR or PR were expressed by >10% of the nuclei in the entire section of the tumour. Tumour Classification
The CMCs were evaluated based on the classification proposed by Goldschmidt et al. (2011) and were subcategorized into three groups: epithelial tumours, special types and mixed tumours. Three histological grades according to Elston and Ellis (1991) were used: grade I, well differentiated; grade II, moderately differentiated; and grade III, poorly differentiated. The cases were also categorized by
Please cite this article in press as: Kim N-H, et al., Evaluation of Clinicopathological Characteristics and Oestrogen Receptor Gene Expression in Oestrogen Receptor-negative, Progesterone..., Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.04.001
Oestrogen Receptor Gene Expression in Canine Mammary Carcinoma
clinicopathological parameters including the presence of central necrosis, lymphatic vessel infiltration, age of the animal and tumour size. Oestrogen Receptor Gene Expression
Levels of OR mRNA were compared in 36 OR/ PR+ tumours, 20 OR/PR tumours and 20 OR+/PR+ tumours. The QuantiGene 2.0 Reagent System was used according to the manufacturer’s protocol. For tissue homogenization, approximately 100e200 mm2 of tumour area, 50 mm thick, was selectively dissected per FFPE sample. Tissue sections were incubated for 5 h with 300 ml of homogenizing solution and 4.5 ml proteinase K in a thermomixer at 65 C and 850 revolutions per minute (Eppendorf, Hamburg, Germany). The homogenates were separated from wax and tissue debris by centrifugation, transferred to a new tube and stored at 70 C until used. Probe set designs for the target gene (canine OR 1; XM_533454) and the reference gene (canine GAPDH; NM_001003142) were requested from Panomics Inc., Fremont, California, USA. To capture the target RNA, the working probe set was prepared in proportion to the number of wells to be run by mixing 33.3 ml of lysis mixture, 25.4 ml nuclease-free water, 1 ml of blocking reagent and 0.3 ml of probe set per well. Forty microlitres of tissue homogenate and 60 ml of working probe set were dispensed into each oligonucleotide-conjugated well of the 96-well capture plate. The plate was incubated for 16 h at 55 C, followed by washing of each well three times with wash buffer. For signal amplification, 100 ml each of 2.0 preamplifier working reagent, 2.0 amplifier working reagent and label probe working reagent were applied consecutively to each well and hybridized for 1 h at 55 C, 55 C and 50 C, respectively. Wells were washed three times with wash buffer between each step. For signal detection, 100 ml of 2.0 substrate was applied to each well and the plate was sealed and incubated at room temperature for 5 min. The luminescence of each well was measured by a luminometer (Turner Biosystems, Sunnyvale, California, USA). To determine assay linearity, each sample was run as a twofold dilution. To normalize the data, the following formula was used: normalized OR ¼ 1,000 (average value for OR average value for background control)/value for canine GAPDH. Statistical Analysis
Pearson’s chi-square test and one-way analysis of variance (ANOVA) with post-hoc test were per-
3
formed to identify the relevance of combined expression of the hormone receptors and clinicopathological parameters: histological type, grade, central necrosis, lymphatic infiltration, age and tumour size. P
