Accepted Manuscript Title: Evidence of epithelial-mesenchymal transition in canine prostate cancer metastasis Author: Carlos Eduardo Fonseca-Alves, Priscilla Emiko Kobayashi, Luis Gabriel Rivera Calderón, Renée Laufer-Amorim PII: DOI: Reference:
S0034-5288(15)00055-7 http://dx.doi.org/doi:10.1016/j.rvsc.2015.03.001 YRVSC 2825
To appear in:
Research in Veterinary Science
Received date: Accepted date:
18-8-2014 1-3-2015
Please cite this article as: Carlos Eduardo Fonseca-Alves, Priscilla Emiko Kobayashi, Luis Gabriel Rivera Calderón, Renée Laufer-Amorim, Evidence of epithelial-mesenchymal transition in canine prostate cancer metastasis, Research in Veterinary Science (2015), http://dx.doi.org/doi:10.1016/j.rvsc.2015.03.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evidence of epithelial-mesenchymal transition in canine prostate cancer metastasis
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Carlos Eduardo Fonseca-Alvesa, Priscilla Emiko Kobayashia, Luis Gabriel Rivera Calderóna,
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Renée Laufer-Amorima
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a
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Estadual Paulista – UNESP, postal code: 18618-970, Botucatu, São Paulo, BRA.
Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, Univ.
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Highlights Normal prostatic tissue was negative for the mesenchymal marker Pre-neoplastic lesions had vimentin-positive cells without loss of epithelial markers EMT occurs in canine prostatic neoplasia, similar to the process in humans
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Abstract
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The epithelial-mesenchymal transition (EMT) is a fundamental event responsible for
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the invasiveness and metastasis of epithelial tumours. The EMT has been described in many
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human cancers, but there are few reports of this phenomenon in veterinary oncology. Due to
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the importance of this process, the current study evaluated mesenchymal and epithelial marker
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protein expression in prostate lesions from dogs. Our results indicate both a loss of E-cadherin
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and translocation of β-catenin from the membrane to the cytoplasm and nucleus in the tumour
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group. Vimentin expression in the tumour group was higher than in normal tissue. All of the
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metastases were positive for prostate-specific antigen, pan-cytokeratin and E-cadherin,
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although fewer positive cells were present than in the primary tumours. The
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immunohistochemical results showed a loss of epithelial markers and a gain of a mesenchymal
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marker among metastatic cells, suggesting that the EMT occurs during the metastatic process
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of canine prostate carcinoma.
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Keywords: Dog; Prostate cancer; Immunohistochemistry; Epithelial-mesenchymal crosstalk
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1. Introduction
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The leading cause of death related to cancer in the Western world is metastasis, which
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represents a final stage of the disease and is associated with a poor prognosis (Pani et al. 2010).
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Death of patients with metastasis occurs due to failure of the affected tissues, concomitant
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paraneoplastic syndromes and metabolic changes (Steeg, 2006). Metastasis is a very complex
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process. Information about metastasis in humans has increased over the years (Pani et al. 2010)
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but is still limited in veterinary medicine.
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Other than humans, dogs are the only species that naturally develops prostatic
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carcinoma (PCa) with high frequency (Fonseca-Alves et al., 2013a; Palmieri et al., 2014). In
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dogs, PCa is highly metastatic, and the disease is usually at an advanced stage at diagnosis
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(Argyle, 2009; Fonseca-Alves et al., 2013b). Many studies from the human cancer literature
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have described a correlation between the epithelial-mesenchymal transition (EMT) and human
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PCa (Frederick et al., 2007; Mani et al., 2008; Thiery et al., 2009; Brabletz et al., 2012). The
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EMT is an important process in the biology of metastasis. During the EMT, epithelial cells lose
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their polarity and cell-cell adhesion function and gain invasive and migratory properties (Mani
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et al., 2008). In this process, the cellular-extracellular matrix interactions and the cellular
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cytoskeleton are modified to confer migratory and invasive abilities (Radisky, 2005). The EMT
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and its converse process, the mesenchymal-epithelial transition (MET), are crucial to the
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carcinogenic process (Frederick et al., 2007).
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The E-cadherin/β-catenin complex plays an important role in cell-to-cell adhesion.
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These molecules are associated with the canonical WNT signalling pathway in human prostate
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cancer (Schmalhofer et al., 2009). E-cadherin and β-catenin are associated with the epithelial
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tissue phenotype and are responsible for regulating cell migration and differentiation (Comijn
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et al., 2001). In the carcinogenic process in epithelial tissues, the absence of these molecules is
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associated with the invasion of adjacent tissues and metastasis implantation (Schmalhofer et al.,
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2009).
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Loss of E-cadherin protein expression is associated with high-grade prostate cancer in
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humans, and these tumours demonstrate a metastatic phenotype (Graff et al., 1995). The
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localisation of β-catenin plays an important role in cell proliferation. When β-catenin is
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localised in the membrane, it is responsible for cell-to-cell adhesion and has an important role
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in the cytoskeleton (Schmalhofer et al., 2009). In the metastatic process of human PCa, cancer
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cells lose E-cadherin and develop β-catenin nuclear staining (Graff et al., 1995). The
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translocation of membranous β-catenin to the nucleus activates the WNT pathway and
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promotes cell proliferation.
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In veterinary medicine, there are few studies on the EMT in PCa. Changes in E-
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cadherin (Fonseca-Alves, et al., 2013a; Rodrigues et al., 2013), β-catenin (Rodrigues et al.,
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2013; Lean et al., 2014) and vimentin (Grieco et al., 2003; Rodrigues et al., 2010; Lean et al.,
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2014) expression were previously described in canine PCa, but these markers have not been
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evaluated in metastatic prostate carcinomas.
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Due to the importance of metastasis to the treatment protocol and survival time of
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patients, this research aimed to verify evidence of the EMT in canine pre-neoplastic prostate
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lesions and in prostate carcinomas and their metastases.
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2. Materials and Methods
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2.1 Animal data
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A total of 36 paraffin blocks from 24 intact adult dogs (aged 1 to 15 years) with a
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clinical history of prostate lesions were obtained from the archives of the Pathology Service at
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Univ. Estadual Paulista. The samples included prostate lesions from five normal prostate cases,
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five benign prostate hyperplasia (BPH) cases, two prostatic intraepithelial neoplasia (PIN)
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cases, three prostatic inflammatory atrophy (PIA) cases, five non-metastatic PCa cases, four
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metastatic PCa cases and 12 metastatic PCa foci cases. To establish the histological diagnoses,
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haematoxylin and eosin (H&E)-stained slides were simultaneously examined by three
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independent pathologists using a multi-head microscope (Leica Microsystems, Germany). The
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diagnoses were made according to published criteria for normal prostate histology (Fonseca-
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Alves et al., 2010), PIN (Cornell et al., 2000), PIA (Fonseca-Alves et al., 2013a) and PCa
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(Palmieri et al., 2014).
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To establish the metastasis status, we included only animals that had undergone a
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thoracic X-ray, an abdominal ultrasound and/or computed tomography (CT) and a necropsy.
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The clinical records of animals with prostate carcinomas were evaluated for clinical signs, the
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survival time and the chemotherapy protocol. In the animals with non-metastatic PCa (3/4), a
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radical prostatectomy was indicated. The primary chemotherapy protocol established for dogs
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without metastasis was carboplatin (300 mg/m2) every 21 days interspersed with doxorubicin
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(30 mg/m2). In dogs that experienced toxicity with this protocol or that had metastasis at
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diagnosis, metronomic chemotherapy with piroxicam (0.3 mg/kg/day) and cyclophosphamide
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(10 mg/m2/day) was established.
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2.2 Immunohistochemistry analysis
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Immunohistochemistry slides were prepared from the paraffin-embedded tissue blocks.
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After deparaffinisation in xylene and graded alcohol, antigen retrieval was performed in
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citrate buffer (pH 6.0) in a pressure cooker (Pascal®; Dako, Carpinteria, CA, USA), and the
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slides were prepared using an automated immunohistochemistry platform (Autostainer
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Classic®, Dako, Carpinteria, USA). The primary antibodies used are listed in Table 1. A
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polymer system (Advance, Dako, Carpinteria, CA, USA) was applied as the secondary
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antibody and was combined with peroxidase. 3, 3’-Diaminobenzidine (DAB) was used as the
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chromogen, and the slides were counterstained with Harry’s haematoxylin.
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Normal canine epithelial prostate tissue and stromal components were used as positive
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internal controls, and the negative control was prepared by replacing the primary antibody with
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Tris buffer.
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Semi-quantitative analysis of pan-cytokeratin, vimentin and E-cadherin expression was
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performed according to a scoring system based on that of Fonseca-Alves et al. (2013a). The
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samples were scored based on an assessment of the number of cells with positive protein
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expression. An absence of positive cells was scored as 0, 1 to 25% positive cells was scored as
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1, 26 to 50% positive cells was scored as 2, 51 to 75% positive cells was scored as 3, and more
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than 75% positive cells was scored 4. The immunohistochemical results were independently
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interpreted by two collaborators. For β-catenin, the location was recorded as the membrane,
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cytoplasm or nucleus, and for prostate-specific antigen (PSA), positive or negative staining
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was recorded.
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2.2 Statistical analysis
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The chi-square test or Fisher’s exact test was used to determine the association between
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the immunohistochemical categorical variables. Kaplan-Meier analysis was used to analyse the
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overall survival time. The statistical analyses were performed using GraphPad Prism 5.0
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software.
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3. Results
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3.1 Clinical data
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Five dogs were diagnosed with BPH associated with dyschezia and tenesmus; five dogs
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were diagnosed with non-metastatic carcinoma associated with dyschezia, tenesmus,
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haematuria and dysuria; and four dogs were diagnosed with metastatic carcinomas. The dogs
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with metastatic carcinomas demonstrated dyschezia, tenesmus, haematuria, dysuria, dyspnoea,
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pain in the lumbosacral region and gait changes in the hind limbs. The most common locations
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of metastatic foci were the lungs (n=4) and bone (sacral region, n=4). Two dogs each had
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metastases in the intestine (n=2) and bladder (n=2) (Table 2). The animals with normal
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prostates (n=5), PIA (n=3) or PIN (n=2) had no clinical symptoms.
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The mean survival time of the dogs with metastasis was 41.25±7.81 days (30 to 60
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days), and that of the dogs without metastasis was 280±87.16 days (30 to 480 days). The dogs
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with metastatic PCa demonstrated a trend towards decreased survival time compared with the
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dogs without metastasis (P=0.0553) (Figure 1).
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The dogs with non-metastatic tumours tolerated the chemotherapy protocol. In total,
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80% (4/5) of the dogs did not present severe side effects, whereas one dog developed severe
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pancytopenia and diarrhoea, so chemotherapy was discontinued. For this dog, metronomic
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therapy was initiated. In addition to this animal, five dogs received metronomic chemotherapy
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(one with a non-metastatic tumour and 4 with metastatic tumours), which was well tolerated by
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all of the animals.
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3.2 Histology
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Among the samples, we found five normal prostate tissue samples, five samples
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showing BPH, two showing PIN, three showing PIA, five showing non-metastatic carcinomas,
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and four showing metastatic carcinomas (Figure 2 A) from 11 different metastatic sites (from
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two dogs with four metastases and the other two with two metastatic foci) (Table 2).
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The different prostate carcinoma samples demonstrated various histologic patterns.
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Among the non-metastatic carcinomas, we identified acinar PCa (n=3) and solid PCa (n=2).
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Among the metastatic PCa cases, we found mixed-pattern (acinar/solid) tumours (n=2), high-
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grade acinar PCa (n=1) and solid PCa (n=1). The main metastatic sites were the lungs (4/4),
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bones (4/4), intestine (2/4) and bladder (1/4) (Table 2). A total of 75% of the tumours (3/4)
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displayed lymphatic neoplastic emboli.
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Of the metastatic tumours that we evaluated, two tumours showed a mixed pattern with
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solid and acinar components. The metastases presented either an acinar or a solid pattern. The
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other two tumours were an acinar carcinoma and a solid carcinoma. The metastases of each
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tumour exhibited the same histological pattern as the primary tumour (Table 2).
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3.3 Immunohistochemistry
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The immunohistochemical results are summarised in Table 2. The epithelial cells from
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the normal prostate tissue and the BPH samples were positive for PSA (Figure 2 D), had a
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score of 4 for E-cadherin and pan-cytokeratin (Figure 2 B), were negative for vimentin (Figure
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2 C), and had membranous β-catenin staining. The prostatic pre-neoplastic lesions of PIN and
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PIA expressed PSA and had a score of 4 for pan-cytokeratin, and less than 25% (score 1) of the
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epithelial cells from the lesions were positive for vimentin. All of these samples had
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membranous β-catenin staining. One PIA sample had score of 1 for E-cadherin (Table 2).
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The non-metastatic PCa samples were positive for PSA and had a score of 4 for pan-
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cytokeratin. All of these samples were positive for vimentin, ranging from a few positive cells
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(less than 25% of all cells, scored as 1) to up to 75% positive among the neoplastic cells. One
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non-metastatic PCa sample was negative for E-cadherin, and all of the samples exhibited
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nuclear and cytoplasmic β-catenin staining. All of the metastatic carcinomas were positive for
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PSA, pan-cytokeratin and vimentin. The metastatic tumours lost E-cadherin expression. Two
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of the metastatic tumours were negative for E-cadherin, and the other two had less than 25%
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positive cells (score 1). β-catenin expression in the metastatic tumours was similar to that in the
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non-metastatic carcinomas (nuclear and cytoplasmic expression).
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The embolic tumour cells were positive for PSA and vimentin and negative for pan-
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cytokeratin and E-cadherin. Several of these cells had cytoplasmic β-catenin expression. All of
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the metastases were positive for PSA, pan-cytokeratin (Figure 3 A) and E-cadherin (Figure 3
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D), although fewer positive cells were present than in the primary tumours. The metastatic
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tumour cells were also positive for vimentin (Figure 3 B), with higher scores than those of the
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non-metastatic tumours but with similar scores to the primary metastatic carcinomas. β-catenin
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staining (Figure 3 C) in the metastatic tumour cells revealed a nuclear and cytoplasmic
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distribution that was similar to that of all of the other prostate carcinomas.
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There was no statistically significant difference in vimentin, cytokeratin, E-cadherin
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and β-catenin staining in the normal samples compared with the metastatic and non-metastatic
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PCa samples, probably due to the low number of samples, although the normal prostatic tissue
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and BPH samples were negative for vimentin. Grouping the normal and BPH samples (n=10)
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together and comparing them with the PCa samples (metastatic and non-metastatic) (n=9), we
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found higher expression of vimentin in the tumours (P=0.042). We did not find a significant
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difference in vimentin staining according the histological tumour pattern (P>0.05), but we did
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observe a trend towards a higher vimentin expression in the tumours with the solid pattern
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compared with the acinar pattern (Table 2).
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Discussion
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Our results demonstrated a trend towards a lower survival time among the dogs with
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metastasis compared with the dogs without metastasis at the time of diagnosis. All of the
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animals with metastatic disease had metastases in the lung and bone, which are common sites
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of metastases in humans (Yonou et al., 2001). Withrow et al. (2013) described a poor prognosis
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for dogs with metastatic PCa: dogs with a diagnosis of PCa without treatment showed a
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survival time of less than 30 days or were euthanised after diagnosis. In veterinary medicine,
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there have been no new therapeutic options proven to be effective for the treatment of PCa. In
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human medicine, new treatments proposed for metastatic PCa have prolonged the survival time
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of patients (Uhlman et al., 2014). These treatments include abiraterone (Bono et al., 2011) and
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a promising prostate cancer vaccine (Uhlman et al., 2014).
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In this study, all of the prostate tissue as well as the metastatic and neoplastic cells in
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the lymph and blood vessels were positive for PSA. Lai et al. (2008) performed
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immunostaining of canine prostate tissue and found high PSA expression in prostate epithelial
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cells. Wu et al. (2014) performed a biochemical characterisation of PSA in cell cultures from
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canine PCa; this study described the expression of PSA in normal and PCa tissues but did not
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evaluate the expression of PSA in metastatic lesions. Our study suggests that PSA is a good
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marker for prostate cells. For example, PSA can be used to identify embolic foci of PSA-
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positive cells in vessels and in metastatic foci. In human medicine, PSA is used as a serum
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biomarker, and PSA immunohistochemistry is used to detect circulating tumour cells (CTCs)
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and to confirm the prostatic origin of metastases (Armstrong et al., 2011).
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The EMT plays a central role in human cancer invasion and metastasis (Geyer et al.
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2013). Vimentin is the main marker for the EMT, showing whether epithelial cells have gained
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a mesenchymal phenotype (Bono et al., 2008). This phenomenon was observed in the canine
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prostatic pre-neoplastic lesions (PIN and PIA) and PCa in the present study. The most
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interesting finding was that the neoplastic embolic cells in the primary tumours exhibited
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changes in all of the protein expression profiles studied, with a loss of epithelial markers and a
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gain of a mesenchymal marker. This pattern was also observed for β-catenin translocation to
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the cytoplasm and nucleus in PCa. Armstrong et al. (2011) evaluated CTCs from human
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patients with metastatic PCa and found co-expression of cytokeratin and vimentin in 100% of
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the CTCs, showing strong evidence that expression of vimentin by circulating epithelial cells is
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important to the EMT phenotype and the metastatic process.
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Another important event in the EMT is loss of E-cadherin (Thiery et al., 2009). In the
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current study, we found E-cadherin loss in the canine pre-neoplastic lesions and PCa and a gain
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of expression of this protein in the metastatic lesions. According to Fonseca-Alves et al.
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(2013a), E-cadherin expression is dynamic: a tumour can lose expression of this protein during
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invasion and metastasis and regain it during cell-cell adhesion and proliferation. According to
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Umbas et al. (1994), the metastatic process in human PCa occurs concurrently with E-cadherin
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loss. Patients negative for immunohistochemical expression of this protein had shorter survival
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times compared with patients with E-cadherin-positive prostate cancer. Patients with advanced
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primary PCa had E-cadherin-, N-cadherin- and O-cadherin-negative CTCs that were positive
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for cytokeratin, independent of cadherin expression (Armstrong et al., 2011). This finding
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suggests that a loss of cadherin proteins is essential for the EMT phenotype and the metastatic
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process in human PCa.
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In the present study, normal prostate tissue was negative for the mesenchymal marker
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vimentin, but the pre-neoplastic lesions contained several vimentin-positive cells, without a
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loss of epithelial markers. Prostate cancer cells with high vimentin expression have an invasive
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phenotype and a greater chance of progression to a metastatic tumour (Rodrigues et al., 2011),
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as shown by the neoplastic emboli in our study. Interestingly, we observed that non-metastatic
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PCa included vimentin- and cytokeratin-positive cells, similar to what was observed in the pre-
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neoplastic lesions, but only metastatic cancer cells had a loss of E-cadherin expression. This
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finding suggests that vimentin expression is related to prostate carcinogenesis in dogs, from the
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development of pre-neoplastic lesions to PCa. However, the loss of E-cadherin contributed to
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malignant potential because only metastatic carcinomas and neoplastic emboli in primary
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tumours showed this pattern of decreased E-cadherin expression.
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In many epithelial cancers, β-catenin gene mutations occur, and the protein localisation
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changes from the membrane to the cytoplasm or nucleus (Geyer et al. 2013). This event was
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evident in canine prostatic metastasis in our study. All of the neoplastic cells from the
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metastatic lesions and the metastatic and non-metastatic PCa showed cytoplasmic expression.
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Lean et al. (2014) showed nuclear/cytoplasmic expression in canine PCa and hypothesised a
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link to the WNT-1 pathway, which induces cell proliferation and tumour progression.
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Canine prostate cancer is considered as an aggressive disease with higher metastasis
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rates and low survival times. Similar to what is observed in humans, in dogs, the transition of
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epithelial cells into the mesenchymal phenotype is associated with cancer aggressiveness, and
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the loss of epithelial markers, such as E-cadherin, is very important for the ability of tumour
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cells to invade. Our results suggest that the metastatic process of spontaneous canine PCa is
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similar to that of human disease. Studying PCa in dogs may help to improve our understanding
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of its metastatic process in both species, and humans may benefit from these studies, including
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future clinical trials using dogs as a natural model for metastatic prostatic disease.
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Conclusions
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The loss of E-cadherin and the translocation of β-catenin from the membrane to the
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cytoplasm/nucleus are important events in the invasion and metastatic processes of canine
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metastatic prostate carcinomas. Vimentin is also important in the carcinogenic process of
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canine prostate carcinomas. In the present study, the normal and hyperplastic tissues were
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negative for vimentin, the pre-neoplastic lesions had the same number of vimentin-positive
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cells as the control, and the tumours had the highest expression. Our immunohistochemical
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findings suggest that the EMT occurs in canine prostate neoplasia and is similar to that in
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humans.
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Conflict of interest statement
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None of the authors of this paper has a financial or personal relationship with other
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people or organisations that could have inappropriately influenced or biased the content of the
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paper.
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Acknowledgements We thank the São Paulo State Research Foundation (FAPESP) for their financial support (Grant No. 2010/13774-6, No. 2012/16426-1, and No. 2012/16068-0).
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Wu LY, Johnson JM, Simmons JK, Mendes DE, Geruntho JJ, Liu T, Dirksen WP, Rosol TJ,
373
Davis WC, Berkman CE., 2014. Biochemical characterization of prostate-specific membrane
374
antigen from canine prostate carcinoma cells. Prostate 74, 451-457.
375 376
Yonou H, Yokose T, Kamijo T, Kanomata N, Hasebe T, Nagai K, Hatano T, Ogawa Y, Ochiai
377
A., 2001. Establishment of a novel species- and tissue-specific metastasis model of human
378
prostate cancer in humanized non-obese diabetic/severe combined immunodeficient mice
379
engrafted with human adult lung and bone. Cancer Research 61, 2177-2182.
380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396
Page 16 of 20
17 397 398 399 400 401 402
Figure 1 – Legend
403
Analysis of the survival time of dogs with metastatic or non-metastatic tumours.
404 405 406 407 408
Figure 2 – Legend
409 410
Photomicrographs showing canine prostate carcinoma. The figure panels show
411
histological alterations (a) and the expression of pan-cytokeratin (b), vimentin (c) and
412
prostate-specific antigen (PSA) (d) in prostate carcinoma. It is possible to note
413
neoplastic proliferation of the prostate epithelial cells (a) with the presence of
414
neoplastic cells in the lymphatic vessels. The expression of pan-cytokeratin can be
415
observed in the neoplastic cells in the epithelium (b), whereas and the neoplastic cells
416
within the lymphatic vessels were negative (arrow). The stromal components and
417
neoplastic cells (arrow) in the lymphatic vessels were positive for vimentin (c). The
418
expression
419
Immunohistochemistry, 3,3’-diaminobenzidine (DAB), counterstaining with Harry’s
420
haematoxylin. Scale bar, 50 μm.
of
PSA
can
be
observed
in
the
prostate
cells
(arrow).
421 422
Page 17 of 20
18 423 424 425
Figure 3 – Legend
426 427
Photomicrographs showing canine metastatic prostate lesions. The figure panels
428
show the expression of pan-cytokeratin (a), vimentin (b), β-catenin (c) and E-cadherin
429
(d) in metastatic prostate foci. It is possible to note the expression of pan-cytokeratin
430
in most of the neoplastic cells (arrowhead) and adjacent intestinal submucosa
431
negative (arrow) (a). Immunohistochemical expression of vimentin in metastatic cells
432
(arrows) with presence of atypical mitosis (circle) (b). The metastatic foci in the
433
intestine show cytoplasmic expression of β-catenin (arrow) (c) and membranous
434
expression of E-cadherin (d). Immunohistochemistry, 3,3’-diaminobenzidine (DAB),
435
counterstaining with Harry’s haematoxylin. Scale bar, 50 μm.
436 437 438
Table 1
439
Antibodies employed in immunnohistochemistry. Antibodies
440 441 442 443 444 445 446
Pan-cytokeratin clone AE1/AE3a Vimentin clone V9a Prostatis Specific antigen (PSA)b E-cadherinb β-cateninc a Dako corporation, Carpinteria, USA. b Invitrogen, Carlsbad, USA. c Spring bioscience, Pleasanton, USA.
Diluition 1:300 1:400 1:1000 1:200 1:200
Table 2. Summary of immunohistochemistry results.
Page 18 of 20
PSA Case number Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Case 11 Case 12 Case 13 Case 14 Case 15 Case 16 Case 17 Case 18 Case 19 Case 20 Case 21 Case 21 Case 21 Case 21 Case 21 Case 22 Case 22 Case 22 Case 22 Case 22 Case 23 Case 23 Case 23 Case 24 Case 24 Case 24 447 448 449 450 451 452 453 454
Diagnosis Normal Normal Normal Normal Normal BPH BPH BPH BPH BPH PIN PIN PIA PIA PIA NPCA NPCA NPCA NPCA NPCA MPCA Lung metastasis Bone metastasis Intestine metastasis Bladder metastasis MPCA Lung metastasis Bone metastasis Intestine metastasis Bladder metastasis MPCA Lung metastasis Bone metastasis MPCA Lung metastasis Bone metastasis
Pancytokeratin
Vimentin
E-cadherin 19
(score*) 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 2 3 4 2 3 3 2 4 3 2 4 3 3
(score*) 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 2 2 3 3 3 2 3 3 2 3 2 3 3 2 3 3 3 2 3 3
(score*) 4 4 4 4 3 4 4 3 4 4 4 4 1 3 3 1 2 2 0 1 0 4 4 3 4 1 2 2 4 3 0 3 2 1 2 3
(+/-) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
nuc nuc nuc nuc nuc nuc
nuc
nuc
nuc
*Score 0; 1 to 25% positive cells received a score of 1; 26 to 50% positive cells, a score of 2; 51 to 75% positive cells, a score of 3; and more than 75% positive cells, a score of 4. BPH: Benign prostatic hyperplasia; PIN: Prostatic intraepithelial neoplasia; PIA: Proliferative inflammatory atrophy; NPCA: non-metastatic prostatic carcinoma; MPCA: metastatic prostatic carcinoma; + positive; - negative.
Page 19 of 20
20 455 456 457 458 459
Page 20 of 20
S0034-5288(15)00055-7 http://dx.doi.org/doi:10.1016/j.rvsc.2015.03.001 YRVSC 2825
To appear in:
Research in Veterinary Science
Received date: Accepted date:
18-8-2014 1-3-2015
Please cite this article as: Carlos Eduardo Fonseca-Alves, Priscilla Emiko Kobayashi, Luis Gabriel Rivera Calderón, Renée Laufer-Amorim, Evidence of epithelial-mesenchymal transition in canine prostate cancer metastasis, Research in Veterinary Science (2015), http://dx.doi.org/doi:10.1016/j.rvsc.2015.03.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 1
Evidence of epithelial-mesenchymal transition in canine prostate cancer metastasis
2 3 4 5
Carlos Eduardo Fonseca-Alvesa, Priscilla Emiko Kobayashia, Luis Gabriel Rivera Calderóna,
6
Renée Laufer-Amorima
7 8
a
9
Estadual Paulista – UNESP, postal code: 18618-970, Botucatu, São Paulo, BRA.
Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, Univ.
10 11 12 13 14 15 16 17 18
Highlights Normal prostatic tissue was negative for the mesenchymal marker Pre-neoplastic lesions had vimentin-positive cells without loss of epithelial markers EMT occurs in canine prostatic neoplasia, similar to the process in humans
Page 1 of 20
2 19
Abstract
20
The epithelial-mesenchymal transition (EMT) is a fundamental event responsible for
21
the invasiveness and metastasis of epithelial tumours. The EMT has been described in many
22
human cancers, but there are few reports of this phenomenon in veterinary oncology. Due to
23
the importance of this process, the current study evaluated mesenchymal and epithelial marker
24
protein expression in prostate lesions from dogs. Our results indicate both a loss of E-cadherin
25
and translocation of β-catenin from the membrane to the cytoplasm and nucleus in the tumour
26
group. Vimentin expression in the tumour group was higher than in normal tissue. All of the
27
metastases were positive for prostate-specific antigen, pan-cytokeratin and E-cadherin,
28
although fewer positive cells were present than in the primary tumours. The
29
immunohistochemical results showed a loss of epithelial markers and a gain of a mesenchymal
30
marker among metastatic cells, suggesting that the EMT occurs during the metastatic process
31
of canine prostate carcinoma.
32 33 34
Keywords: Dog; Prostate cancer; Immunohistochemistry; Epithelial-mesenchymal crosstalk
35
Page 2 of 20
3 36
1. Introduction
37
The leading cause of death related to cancer in the Western world is metastasis, which
38
represents a final stage of the disease and is associated with a poor prognosis (Pani et al. 2010).
39
Death of patients with metastasis occurs due to failure of the affected tissues, concomitant
40
paraneoplastic syndromes and metabolic changes (Steeg, 2006). Metastasis is a very complex
41
process. Information about metastasis in humans has increased over the years (Pani et al. 2010)
42
but is still limited in veterinary medicine.
43
Other than humans, dogs are the only species that naturally develops prostatic
44
carcinoma (PCa) with high frequency (Fonseca-Alves et al., 2013a; Palmieri et al., 2014). In
45
dogs, PCa is highly metastatic, and the disease is usually at an advanced stage at diagnosis
46
(Argyle, 2009; Fonseca-Alves et al., 2013b). Many studies from the human cancer literature
47
have described a correlation between the epithelial-mesenchymal transition (EMT) and human
48
PCa (Frederick et al., 2007; Mani et al., 2008; Thiery et al., 2009; Brabletz et al., 2012). The
49
EMT is an important process in the biology of metastasis. During the EMT, epithelial cells lose
50
their polarity and cell-cell adhesion function and gain invasive and migratory properties (Mani
51
et al., 2008). In this process, the cellular-extracellular matrix interactions and the cellular
52
cytoskeleton are modified to confer migratory and invasive abilities (Radisky, 2005). The EMT
53
and its converse process, the mesenchymal-epithelial transition (MET), are crucial to the
54
carcinogenic process (Frederick et al., 2007).
55
The E-cadherin/β-catenin complex plays an important role in cell-to-cell adhesion.
56
These molecules are associated with the canonical WNT signalling pathway in human prostate
57
cancer (Schmalhofer et al., 2009). E-cadherin and β-catenin are associated with the epithelial
58
tissue phenotype and are responsible for regulating cell migration and differentiation (Comijn
59
et al., 2001). In the carcinogenic process in epithelial tissues, the absence of these molecules is
60
associated with the invasion of adjacent tissues and metastasis implantation (Schmalhofer et al.,
61
2009).
Page 3 of 20
4 62
Loss of E-cadherin protein expression is associated with high-grade prostate cancer in
63
humans, and these tumours demonstrate a metastatic phenotype (Graff et al., 1995). The
64
localisation of β-catenin plays an important role in cell proliferation. When β-catenin is
65
localised in the membrane, it is responsible for cell-to-cell adhesion and has an important role
66
in the cytoskeleton (Schmalhofer et al., 2009). In the metastatic process of human PCa, cancer
67
cells lose E-cadherin and develop β-catenin nuclear staining (Graff et al., 1995). The
68
translocation of membranous β-catenin to the nucleus activates the WNT pathway and
69
promotes cell proliferation.
70
In veterinary medicine, there are few studies on the EMT in PCa. Changes in E-
71
cadherin (Fonseca-Alves, et al., 2013a; Rodrigues et al., 2013), β-catenin (Rodrigues et al.,
72
2013; Lean et al., 2014) and vimentin (Grieco et al., 2003; Rodrigues et al., 2010; Lean et al.,
73
2014) expression were previously described in canine PCa, but these markers have not been
74
evaluated in metastatic prostate carcinomas.
75
Due to the importance of metastasis to the treatment protocol and survival time of
76
patients, this research aimed to verify evidence of the EMT in canine pre-neoplastic prostate
77
lesions and in prostate carcinomas and their metastases.
78 79
2. Materials and Methods
80
2.1 Animal data
81
A total of 36 paraffin blocks from 24 intact adult dogs (aged 1 to 15 years) with a
82
clinical history of prostate lesions were obtained from the archives of the Pathology Service at
83
Univ. Estadual Paulista. The samples included prostate lesions from five normal prostate cases,
84
five benign prostate hyperplasia (BPH) cases, two prostatic intraepithelial neoplasia (PIN)
85
cases, three prostatic inflammatory atrophy (PIA) cases, five non-metastatic PCa cases, four
86
metastatic PCa cases and 12 metastatic PCa foci cases. To establish the histological diagnoses,
87
haematoxylin and eosin (H&E)-stained slides were simultaneously examined by three
Page 4 of 20
5 88
independent pathologists using a multi-head microscope (Leica Microsystems, Germany). The
89
diagnoses were made according to published criteria for normal prostate histology (Fonseca-
90
Alves et al., 2010), PIN (Cornell et al., 2000), PIA (Fonseca-Alves et al., 2013a) and PCa
91
(Palmieri et al., 2014).
92
To establish the metastasis status, we included only animals that had undergone a
93
thoracic X-ray, an abdominal ultrasound and/or computed tomography (CT) and a necropsy.
94
The clinical records of animals with prostate carcinomas were evaluated for clinical signs, the
95
survival time and the chemotherapy protocol. In the animals with non-metastatic PCa (3/4), a
96
radical prostatectomy was indicated. The primary chemotherapy protocol established for dogs
97
without metastasis was carboplatin (300 mg/m2) every 21 days interspersed with doxorubicin
98
(30 mg/m2). In dogs that experienced toxicity with this protocol or that had metastasis at
99
diagnosis, metronomic chemotherapy with piroxicam (0.3 mg/kg/day) and cyclophosphamide
100
(10 mg/m2/day) was established.
101 102
2.2 Immunohistochemistry analysis
103
Immunohistochemistry slides were prepared from the paraffin-embedded tissue blocks.
104
After deparaffinisation in xylene and graded alcohol, antigen retrieval was performed in
105
citrate buffer (pH 6.0) in a pressure cooker (Pascal®; Dako, Carpinteria, CA, USA), and the
106
slides were prepared using an automated immunohistochemistry platform (Autostainer
107
Classic®, Dako, Carpinteria, USA). The primary antibodies used are listed in Table 1. A
108
polymer system (Advance, Dako, Carpinteria, CA, USA) was applied as the secondary
109
antibody and was combined with peroxidase. 3, 3’-Diaminobenzidine (DAB) was used as the
110
chromogen, and the slides were counterstained with Harry’s haematoxylin.
111
Normal canine epithelial prostate tissue and stromal components were used as positive
112
internal controls, and the negative control was prepared by replacing the primary antibody with
113
Tris buffer.
Page 5 of 20
6 114
Semi-quantitative analysis of pan-cytokeratin, vimentin and E-cadherin expression was
115
performed according to a scoring system based on that of Fonseca-Alves et al. (2013a). The
116
samples were scored based on an assessment of the number of cells with positive protein
117
expression. An absence of positive cells was scored as 0, 1 to 25% positive cells was scored as
118
1, 26 to 50% positive cells was scored as 2, 51 to 75% positive cells was scored as 3, and more
119
than 75% positive cells was scored 4. The immunohistochemical results were independently
120
interpreted by two collaborators. For β-catenin, the location was recorded as the membrane,
121
cytoplasm or nucleus, and for prostate-specific antigen (PSA), positive or negative staining
122
was recorded.
123 124
2.2 Statistical analysis
125
The chi-square test or Fisher’s exact test was used to determine the association between
126
the immunohistochemical categorical variables. Kaplan-Meier analysis was used to analyse the
127
overall survival time. The statistical analyses were performed using GraphPad Prism 5.0
128
software.
129 130
3. Results
131
3.1 Clinical data
132
Five dogs were diagnosed with BPH associated with dyschezia and tenesmus; five dogs
133
were diagnosed with non-metastatic carcinoma associated with dyschezia, tenesmus,
134
haematuria and dysuria; and four dogs were diagnosed with metastatic carcinomas. The dogs
135
with metastatic carcinomas demonstrated dyschezia, tenesmus, haematuria, dysuria, dyspnoea,
136
pain in the lumbosacral region and gait changes in the hind limbs. The most common locations
137
of metastatic foci were the lungs (n=4) and bone (sacral region, n=4). Two dogs each had
138
metastases in the intestine (n=2) and bladder (n=2) (Table 2). The animals with normal
139
prostates (n=5), PIA (n=3) or PIN (n=2) had no clinical symptoms.
Page 6 of 20
7 140
The mean survival time of the dogs with metastasis was 41.25±7.81 days (30 to 60
141
days), and that of the dogs without metastasis was 280±87.16 days (30 to 480 days). The dogs
142
with metastatic PCa demonstrated a trend towards decreased survival time compared with the
143
dogs without metastasis (P=0.0553) (Figure 1).
144
The dogs with non-metastatic tumours tolerated the chemotherapy protocol. In total,
145
80% (4/5) of the dogs did not present severe side effects, whereas one dog developed severe
146
pancytopenia and diarrhoea, so chemotherapy was discontinued. For this dog, metronomic
147
therapy was initiated. In addition to this animal, five dogs received metronomic chemotherapy
148
(one with a non-metastatic tumour and 4 with metastatic tumours), which was well tolerated by
149
all of the animals.
150 151
3.2 Histology
152
Among the samples, we found five normal prostate tissue samples, five samples
153
showing BPH, two showing PIN, three showing PIA, five showing non-metastatic carcinomas,
154
and four showing metastatic carcinomas (Figure 2 A) from 11 different metastatic sites (from
155
two dogs with four metastases and the other two with two metastatic foci) (Table 2).
156
The different prostate carcinoma samples demonstrated various histologic patterns.
157
Among the non-metastatic carcinomas, we identified acinar PCa (n=3) and solid PCa (n=2).
158
Among the metastatic PCa cases, we found mixed-pattern (acinar/solid) tumours (n=2), high-
159
grade acinar PCa (n=1) and solid PCa (n=1). The main metastatic sites were the lungs (4/4),
160
bones (4/4), intestine (2/4) and bladder (1/4) (Table 2). A total of 75% of the tumours (3/4)
161
displayed lymphatic neoplastic emboli.
162
Of the metastatic tumours that we evaluated, two tumours showed a mixed pattern with
163
solid and acinar components. The metastases presented either an acinar or a solid pattern. The
164
other two tumours were an acinar carcinoma and a solid carcinoma. The metastases of each
165
tumour exhibited the same histological pattern as the primary tumour (Table 2).
Page 7 of 20
8 166 167
3.3 Immunohistochemistry
168
The immunohistochemical results are summarised in Table 2. The epithelial cells from
169
the normal prostate tissue and the BPH samples were positive for PSA (Figure 2 D), had a
170
score of 4 for E-cadherin and pan-cytokeratin (Figure 2 B), were negative for vimentin (Figure
171
2 C), and had membranous β-catenin staining. The prostatic pre-neoplastic lesions of PIN and
172
PIA expressed PSA and had a score of 4 for pan-cytokeratin, and less than 25% (score 1) of the
173
epithelial cells from the lesions were positive for vimentin. All of these samples had
174
membranous β-catenin staining. One PIA sample had score of 1 for E-cadherin (Table 2).
175
The non-metastatic PCa samples were positive for PSA and had a score of 4 for pan-
176
cytokeratin. All of these samples were positive for vimentin, ranging from a few positive cells
177
(less than 25% of all cells, scored as 1) to up to 75% positive among the neoplastic cells. One
178
non-metastatic PCa sample was negative for E-cadherin, and all of the samples exhibited
179
nuclear and cytoplasmic β-catenin staining. All of the metastatic carcinomas were positive for
180
PSA, pan-cytokeratin and vimentin. The metastatic tumours lost E-cadherin expression. Two
181
of the metastatic tumours were negative for E-cadherin, and the other two had less than 25%
182
positive cells (score 1). β-catenin expression in the metastatic tumours was similar to that in the
183
non-metastatic carcinomas (nuclear and cytoplasmic expression).
184
The embolic tumour cells were positive for PSA and vimentin and negative for pan-
185
cytokeratin and E-cadherin. Several of these cells had cytoplasmic β-catenin expression. All of
186
the metastases were positive for PSA, pan-cytokeratin (Figure 3 A) and E-cadherin (Figure 3
187
D), although fewer positive cells were present than in the primary tumours. The metastatic
188
tumour cells were also positive for vimentin (Figure 3 B), with higher scores than those of the
189
non-metastatic tumours but with similar scores to the primary metastatic carcinomas. β-catenin
190
staining (Figure 3 C) in the metastatic tumour cells revealed a nuclear and cytoplasmic
191
distribution that was similar to that of all of the other prostate carcinomas.
Page 8 of 20
9 192
There was no statistically significant difference in vimentin, cytokeratin, E-cadherin
193
and β-catenin staining in the normal samples compared with the metastatic and non-metastatic
194
PCa samples, probably due to the low number of samples, although the normal prostatic tissue
195
and BPH samples were negative for vimentin. Grouping the normal and BPH samples (n=10)
196
together and comparing them with the PCa samples (metastatic and non-metastatic) (n=9), we
197
found higher expression of vimentin in the tumours (P=0.042). We did not find a significant
198
difference in vimentin staining according the histological tumour pattern (P>0.05), but we did
199
observe a trend towards a higher vimentin expression in the tumours with the solid pattern
200
compared with the acinar pattern (Table 2).
201 202
Discussion
203
Our results demonstrated a trend towards a lower survival time among the dogs with
204
metastasis compared with the dogs without metastasis at the time of diagnosis. All of the
205
animals with metastatic disease had metastases in the lung and bone, which are common sites
206
of metastases in humans (Yonou et al., 2001). Withrow et al. (2013) described a poor prognosis
207
for dogs with metastatic PCa: dogs with a diagnosis of PCa without treatment showed a
208
survival time of less than 30 days or were euthanised after diagnosis. In veterinary medicine,
209
there have been no new therapeutic options proven to be effective for the treatment of PCa. In
210
human medicine, new treatments proposed for metastatic PCa have prolonged the survival time
211
of patients (Uhlman et al., 2014). These treatments include abiraterone (Bono et al., 2011) and
212
a promising prostate cancer vaccine (Uhlman et al., 2014).
213
In this study, all of the prostate tissue as well as the metastatic and neoplastic cells in
214
the lymph and blood vessels were positive for PSA. Lai et al. (2008) performed
215
immunostaining of canine prostate tissue and found high PSA expression in prostate epithelial
216
cells. Wu et al. (2014) performed a biochemical characterisation of PSA in cell cultures from
217
canine PCa; this study described the expression of PSA in normal and PCa tissues but did not
Page 9 of 20
10 218
evaluate the expression of PSA in metastatic lesions. Our study suggests that PSA is a good
219
marker for prostate cells. For example, PSA can be used to identify embolic foci of PSA-
220
positive cells in vessels and in metastatic foci. In human medicine, PSA is used as a serum
221
biomarker, and PSA immunohistochemistry is used to detect circulating tumour cells (CTCs)
222
and to confirm the prostatic origin of metastases (Armstrong et al., 2011).
223
The EMT plays a central role in human cancer invasion and metastasis (Geyer et al.
224
2013). Vimentin is the main marker for the EMT, showing whether epithelial cells have gained
225
a mesenchymal phenotype (Bono et al., 2008). This phenomenon was observed in the canine
226
prostatic pre-neoplastic lesions (PIN and PIA) and PCa in the present study. The most
227
interesting finding was that the neoplastic embolic cells in the primary tumours exhibited
228
changes in all of the protein expression profiles studied, with a loss of epithelial markers and a
229
gain of a mesenchymal marker. This pattern was also observed for β-catenin translocation to
230
the cytoplasm and nucleus in PCa. Armstrong et al. (2011) evaluated CTCs from human
231
patients with metastatic PCa and found co-expression of cytokeratin and vimentin in 100% of
232
the CTCs, showing strong evidence that expression of vimentin by circulating epithelial cells is
233
important to the EMT phenotype and the metastatic process.
234
Another important event in the EMT is loss of E-cadherin (Thiery et al., 2009). In the
235
current study, we found E-cadherin loss in the canine pre-neoplastic lesions and PCa and a gain
236
of expression of this protein in the metastatic lesions. According to Fonseca-Alves et al.
237
(2013a), E-cadherin expression is dynamic: a tumour can lose expression of this protein during
238
invasion and metastasis and regain it during cell-cell adhesion and proliferation. According to
239
Umbas et al. (1994), the metastatic process in human PCa occurs concurrently with E-cadherin
240
loss. Patients negative for immunohistochemical expression of this protein had shorter survival
241
times compared with patients with E-cadherin-positive prostate cancer. Patients with advanced
242
primary PCa had E-cadherin-, N-cadherin- and O-cadherin-negative CTCs that were positive
243
for cytokeratin, independent of cadherin expression (Armstrong et al., 2011). This finding
Page 10 of 20
11 244
suggests that a loss of cadherin proteins is essential for the EMT phenotype and the metastatic
245
process in human PCa.
246
In the present study, normal prostate tissue was negative for the mesenchymal marker
247
vimentin, but the pre-neoplastic lesions contained several vimentin-positive cells, without a
248
loss of epithelial markers. Prostate cancer cells with high vimentin expression have an invasive
249
phenotype and a greater chance of progression to a metastatic tumour (Rodrigues et al., 2011),
250
as shown by the neoplastic emboli in our study. Interestingly, we observed that non-metastatic
251
PCa included vimentin- and cytokeratin-positive cells, similar to what was observed in the pre-
252
neoplastic lesions, but only metastatic cancer cells had a loss of E-cadherin expression. This
253
finding suggests that vimentin expression is related to prostate carcinogenesis in dogs, from the
254
development of pre-neoplastic lesions to PCa. However, the loss of E-cadherin contributed to
255
malignant potential because only metastatic carcinomas and neoplastic emboli in primary
256
tumours showed this pattern of decreased E-cadherin expression.
257
In many epithelial cancers, β-catenin gene mutations occur, and the protein localisation
258
changes from the membrane to the cytoplasm or nucleus (Geyer et al. 2013). This event was
259
evident in canine prostatic metastasis in our study. All of the neoplastic cells from the
260
metastatic lesions and the metastatic and non-metastatic PCa showed cytoplasmic expression.
261
Lean et al. (2014) showed nuclear/cytoplasmic expression in canine PCa and hypothesised a
262
link to the WNT-1 pathway, which induces cell proliferation and tumour progression.
263
Canine prostate cancer is considered as an aggressive disease with higher metastasis
264
rates and low survival times. Similar to what is observed in humans, in dogs, the transition of
265
epithelial cells into the mesenchymal phenotype is associated with cancer aggressiveness, and
266
the loss of epithelial markers, such as E-cadherin, is very important for the ability of tumour
267
cells to invade. Our results suggest that the metastatic process of spontaneous canine PCa is
268
similar to that of human disease. Studying PCa in dogs may help to improve our understanding
Page 11 of 20
12 269
of its metastatic process in both species, and humans may benefit from these studies, including
270
future clinical trials using dogs as a natural model for metastatic prostatic disease.
271 272
Conclusions
273
The loss of E-cadherin and the translocation of β-catenin from the membrane to the
274
cytoplasm/nucleus are important events in the invasion and metastatic processes of canine
275
metastatic prostate carcinomas. Vimentin is also important in the carcinogenic process of
276
canine prostate carcinomas. In the present study, the normal and hyperplastic tissues were
277
negative for vimentin, the pre-neoplastic lesions had the same number of vimentin-positive
278
cells as the control, and the tumours had the highest expression. Our immunohistochemical
279
findings suggest that the EMT occurs in canine prostate neoplasia and is similar to that in
280
humans.
281 282
Conflict of interest statement
283
None of the authors of this paper has a financial or personal relationship with other
284
people or organisations that could have inappropriately influenced or biased the content of the
285
paper.
286 287 288 289
Acknowledgements We thank the São Paulo State Research Foundation (FAPESP) for their financial support (Grant No. 2010/13774-6, No. 2012/16426-1, and No. 2012/16068-0).
290 291
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Figure 1 – Legend
403
Analysis of the survival time of dogs with metastatic or non-metastatic tumours.
404 405 406 407 408
Figure 2 – Legend
409 410
Photomicrographs showing canine prostate carcinoma. The figure panels show
411
histological alterations (a) and the expression of pan-cytokeratin (b), vimentin (c) and
412
prostate-specific antigen (PSA) (d) in prostate carcinoma. It is possible to note
413
neoplastic proliferation of the prostate epithelial cells (a) with the presence of
414
neoplastic cells in the lymphatic vessels. The expression of pan-cytokeratin can be
415
observed in the neoplastic cells in the epithelium (b), whereas and the neoplastic cells
416
within the lymphatic vessels were negative (arrow). The stromal components and
417
neoplastic cells (arrow) in the lymphatic vessels were positive for vimentin (c). The
418
expression
419
Immunohistochemistry, 3,3’-diaminobenzidine (DAB), counterstaining with Harry’s
420
haematoxylin. Scale bar, 50 μm.
of
PSA
can
be
observed
in
the
prostate
cells
(arrow).
421 422
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18 423 424 425
Figure 3 – Legend
426 427
Photomicrographs showing canine metastatic prostate lesions. The figure panels
428
show the expression of pan-cytokeratin (a), vimentin (b), β-catenin (c) and E-cadherin
429
(d) in metastatic prostate foci. It is possible to note the expression of pan-cytokeratin
430
in most of the neoplastic cells (arrowhead) and adjacent intestinal submucosa
431
negative (arrow) (a). Immunohistochemical expression of vimentin in metastatic cells
432
(arrows) with presence of atypical mitosis (circle) (b). The metastatic foci in the
433
intestine show cytoplasmic expression of β-catenin (arrow) (c) and membranous
434
expression of E-cadherin (d). Immunohistochemistry, 3,3’-diaminobenzidine (DAB),
435
counterstaining with Harry’s haematoxylin. Scale bar, 50 μm.
436 437 438
Table 1
439
Antibodies employed in immunnohistochemistry. Antibodies
440 441 442 443 444 445 446
Pan-cytokeratin clone AE1/AE3a Vimentin clone V9a Prostatis Specific antigen (PSA)b E-cadherinb β-cateninc a Dako corporation, Carpinteria, USA. b Invitrogen, Carlsbad, USA. c Spring bioscience, Pleasanton, USA.
Diluition 1:300 1:400 1:1000 1:200 1:200
Table 2. Summary of immunohistochemistry results.
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PSA Case number Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Case 11 Case 12 Case 13 Case 14 Case 15 Case 16 Case 17 Case 18 Case 19 Case 20 Case 21 Case 21 Case 21 Case 21 Case 21 Case 22 Case 22 Case 22 Case 22 Case 22 Case 23 Case 23 Case 23 Case 24 Case 24 Case 24 447 448 449 450 451 452 453 454
Diagnosis Normal Normal Normal Normal Normal BPH BPH BPH BPH BPH PIN PIN PIA PIA PIA NPCA NPCA NPCA NPCA NPCA MPCA Lung metastasis Bone metastasis Intestine metastasis Bladder metastasis MPCA Lung metastasis Bone metastasis Intestine metastasis Bladder metastasis MPCA Lung metastasis Bone metastasis MPCA Lung metastasis Bone metastasis
Pancytokeratin
Vimentin
E-cadherin 19
(score*) 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 2 3 4 2 3 3 2 4 3 2 4 3 3
(score*) 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 2 2 3 3 3 2 3 3 2 3 2 3 3 2 3 3 3 2 3 3
(score*) 4 4 4 4 3 4 4 3 4 4 4 4 1 3 3 1 2 2 0 1 0 4 4 3 4 1 2 2 4 3 0 3 2 1 2 3
(+/-) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
nuc nuc nuc nuc nuc nuc
nuc
nuc
nuc
*Score 0; 1 to 25% positive cells received a score of 1; 26 to 50% positive cells, a score of 2; 51 to 75% positive cells, a score of 3; and more than 75% positive cells, a score of 4. BPH: Benign prostatic hyperplasia; PIN: Prostatic intraepithelial neoplasia; PIA: Proliferative inflammatory atrophy; NPCA: non-metastatic prostatic carcinoma; MPCA: metastatic prostatic carcinoma; + positive; - negative.
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