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Sarcomatoid renal cell carcinoma is an example of epithelial–mesenchymal transition
  1. Joanna L Conant1,
  2. Zhihua Peng2,
  3. Mark F Evans2,
  4. Shelly Naud3,
  5. Kumarasen Cooper2
  1. 1University of Vermont College of Medicine, Burlington, Vermont, USA
  2. 2Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont, USA
  3. 3Medical Biostatistics, University of Vermont College of Medicine, Burlington, Vermont, USA
  1. Correspondence to Joanna Conant, University of Vermont College of Medicine, 89 Beaumont Avenue, Given Box B19, Burlington, VT 05405, USA; joanna.conant{at}uvm.edu

Abstract

Background Sarcomatoid renal cell carcinomas (SRCC) are composed of two cell populations, a sarcomatous component (SC) and a carcinomatous component (CC). SRCC are particularly aggressive and often present at an advanced stage at diagnosis. Epithelial–mesenchymal transition (EMT) has been proposed as a mechanism for the development of SC from CC.

Aims and methods E- to N-cadherin switching, localisation of β-catenin, and expression of Snail and secreted protein acidic and rich in cysteine (SPARC) (markers of EMT) were studied to determine whether SRCC is an example of EMT. Expression of these markers was analysed by immunohistochemistry on 21 cases of SRCC that had both SC and CC and scored according to intensity and extent.

Results E-cadherin expression was decreased in SC (Wilcoxon signed-rank test, p=0.0004) while N-cadherin expression was high in both components (p=0.46). Membranous β-catenin expression was decreased in SC (p<0.0001) while cytoplasmic expression was increased (p=0.0002). Snail and SPARC had higher expression in SC (p=0.002 and p<0.0001, respectively). When the scores were dichotomised into low and high expression levels, the results using McNemar's test substantiated the above results.

Conclusions E- to N-cadherin switching, dissociation of β-catenin from the membrane, and increased expression of Snail and SPARC in SC indicate that SRCC is an example of EMT. High expression of N-cadherin and Snail in CC suggest early involvement in initiating EMT. Once EMT is established, loss of E-cadherin, release of β-catenin into the cytoplasm, and expression of SPARC correspond with mesenchymal phenotypic expression.

  • Renal cell carcinoma
  • epithelial–mesenchymal transition
  • immunohistochemistry
  • neoplasms
  • surgical pathology
  • methodology
  • in situ hybridisation
  • molecular pathology
  • fish
  • HPV
  • cervical cancer
  • papilloma viruses
  • gynaecological pathology
  • soft tissue tumours
  • histopathology
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Introduction

Renal cell carcinoma (RCC) is the most common type of primary cancer affecting the kidneys, accounting for 80% of cases. Occasionally, RCC have sarcomatoid features and these are known as sarcomatoid renal cell carcinomas (SRCC). In SRCC, the tumour is composed of two different cell populations: epithelial cells (the carcinoma component, CC) and mesenchymal cells (the sarcomatoid component, SC). SRCC can be found in all subtypes of RCC, but is most common in chromophobe and clear cell subtypes.1 When sarcomatoid features are present, the tumour is particularly aggressive and therefore present at an advanced stage at diagnosis, with a higher propensity to metastasise.1–3

Evidence suggests that SC arise from CC as a consequence of clonal expansion of a sub-population of neoplastic cells, and that the mesenchymal cells in SC have undergone a morphological modification from their epithelial origin.2 4 We hypothesised that the mesenchymal cells in SRCC have undergone this process through the mechanism known as epithelial–mesenchymal transition (EMT). In EMT, epithelial cells lose their phenotypic epithelial characteristics and gain mesenchymal characteristics, leading to an increased ability to migrate and metastasise.5 It is the acquisition of these mesenchymal capabilities that enables SRCC to be more aggressive.

Cells that have undergone EMT may be evaluated by examining the expression of biomarkers using immunohistochemical staining.6 In this study, we examined both epithelial and mesenchymal markers and proposed a loss of epithelial markers with a gain of mesenchymal markers in SC compared to CC.

In many carcinomas, EMT-inducing transcription factors such as Snail, Slug and Twist are triggered by signals (eg, TGF-β) originating from the tumour-associated stroma. These transcription factors begin the EMT process via intracellular signalling networks involving the proteins β-catenin, MAPK, Smads, Ras and others.7

Loss of E-cadherin and gain of N-cadherin is associated with increased invasive potential in carcinomas and this E- to N-cadherin switch is a hallmark of EMT.5 Normal E-cadherin expression plays a vital role in the maintenance of epithelial integrity and polarity, and is involved in the establishment of cell–cell adhesion.3 8 9 Loss of E-cadherin expression is associated with tumour dedifferentiation, invasion and metastasis in a variety of carcinomas, including oesophagus,10 11 breast,12 prostate13 and lung carcinoma.14

E-cadherin is normally linked to the cytoskeleton by β-catenin; proper linkage between the two is necessary for intercellular connections.3 9 15 When cells undergo EMT, β-catenin dissociates from the membrane, accumulates in the cytoplasm, and finally aggregates into the nucleus, where it acts as a transcriptional activator of Snail, a major E-cadherin transcriptional repressor.5 7 16 The up-regulation of N-cadherin, which is normally expressed in mesenchymal cells, is also associated with EMT in carcinomas such as pancreas,17 breast,18 and head and neck.19

Finally, secreted protein acidic and rich in cysteine (SPARC) is a secreted glycoprotein that modifies cell–extracellular matrix interactions during proliferation and extracellular remodelling, allowing for increased motility. SPARC may regulate neovascularisation and tumour invasion, and has been found to be up-regulated in EMT.20

The purpose of this study was to investigate whether SRCC is an example of EMT by evaluating the loss of epithelial markers in conjunction with the gain of mesenchymal markers in SRCC. To this end, expression of E- and N-cadherin, β-catenin, Snail and SPARC were immunohistochemically examined in a cohort of SRCC having both SC and CC components.

Materials and methods

After this study was approved by the University of Vermont Institutional Review Board and Vermont Cancer Center Protocol Review Committee, a search was made of Fletcher Allen Health Care archives for cases of SRCC resected between 1998 and 2010. A total of 24 cases of SRCC were identified. Cases were selected for inclusion in the study if they comprised morphologically recognisable RCC with definite SC. Cases without CC were excluded since the intention was to compare SC to CC within each individual tumour; 21 cases met the selection criteria.

H&E stained slides were reviewed to confirm the diagnosis, and formalin-fixed, paraffin-embedded tissue blocks were selected from each case for immunohistochemical study; blocks represented SC and CC. Sections (5 μm thick) were cut, deparaffinised in xylene, and rehydrated through a graded series of ethanol solutions, and then washed with deionized (DI) water. For Snail (1:1000 dilution, rabbit polyclonal anti-Snail (ab85931), Abcam, Cambridge, Massachusetts, USA), E-cadherin (1:50 dilution, mouse monoclonal anti-E-cadherin (NCH-38), Dako North America, Carpinteria, California, USA), N-cadherin (1:400 dilution, rabbit polyclonal anti-N-cadherin (ab12221), Abcam), and SPARC (1:50 dilution, rabbit polyclonal anti-SPARC (HPA002989), Sigma-Aldrich Co., St Louis, Missouri, USA), heat induced epitope retrieval was performed by immersion of tissue sections in a water bath at 98°C for 20 min in a 10 mM sodium citrate solution at a pH of 6.0. For β-catenin (1:2000 dilution, mouse anti-β-catenin (14/β-catenin), BD Transduction Laboratories, Franklin Lakes, New Jersey, USA), heat induced epitope retrieval was performed with citrate pH 6.0 solution using a Decloaking Chamber pressure cooker (Biocare Medical, Concord, California, USA) at 100°C for 10 min. Following antigen retrieval, the tissue sections were allowed to cool in solution for 20 min and then rinsed with Tris-buffered saline. Endogenous peroxidase was blocked in 3% hydrogen peroxide in methanol for 15 min and then rinsed with Tris-buffered saline. All antibodies except SPARC were incubated for 30 min at room temperature; SPARC antibody was incubated for 45 min. All antibodies were diluted in Dako antibody diluent (Dako North America). All slides were stained with Dako Autostainer Universal Staining System DC-3400 (Dako North America). The sections were then counterstained with haematoxylin and mounted. All slides for each biomarker were stained in the same batch to ensure consistency of staining intensity. Positive and negative control staining yielded appropriate results.

The stained slides were reviewed by two of the authors (JLC, KC). One author reviewed all slides and the second author reviewed equivocal slides with consensus achieved. The second author also reviewed random slides to ensure consistency. A semi-quantitative estimation based on intensity and extent of stained cells of SC and CC for each case was performed. The intensity was graded from 0 to 3 (0=negative, 1=mild, 2=moderate, 3=strong). The extent was graded from 0 to 3 (0=<5%, 1=5–25%, 2=26–75%, 3=>75%). The intensity and extent scores were summed to obtain the composite score (0–6). A score ≤3 was considered low expression and a score >3 was considered high expression. The Wilcoxon signed-rank test was used to analyse the composite score of SC to CC within individual cases. The scores were then dichotomised into low and high expression levels and analysed using McNemar's test.

Results

Clinicopathological findings

The clinicopathological data are presented in table 1. Radical nephrectomy was performed in all cases. The CC were categorised into clear cell, chromophobe, papillary, and RCC not otherwise specified. The SC were composed of pleomorphic spindle-shaped or epitheloid cells with large nuclei (figure 1). Mean age was 59.8 years and average size of the tumour was 10.0 cm.

Table 1

Clinicopathological data

Figure 1

Light micrograph illustrating sarcomatoid component (A) and carcinoma component (B). H&E stain, 20× objective.

Immunohistochemical findings

The immunohistochemistry data are presented in table 2. Nuclear expression of Snail, cytoplasmic expression of β-catenin, and cytoplasmic expression of SPARC were significantly increased in SC compared to CC when evaluating composite scores (Wilcoxon signed-rank test, figures 2–4). Membranous expression of both β-catenin and E-cadherin was significantly decreased in SC (Wilcoxon signed-rank test, figures 3 and 5). The expression of N-cadherin was similar, but localisation was more often cytoplasmic in SC and membranous in CC (McNemar's test, p=0.02, figure 6). When the scores were dichotomised into low and high expression levels, the results using McNemar's test substantiated these results (p=0.046 for Snail, p=0.004 for E-cadherin, p=1.000 for N-cadherin, p<0.0001 for membranous β-catenin, p=0.03 for cytoplasmic β-catenin, and p=0.0009 for SPARC).

Table 2

Mean expression in SC and CC

Figure 2

Snail, 20× objective. (A) Diffuse nuclear expression in sarcomatoid component, composite score 5.7. (B) Diffuse nuclear expression in carcinoma component, composite score 4.7.

Figure 3

β-catenin, 20× objective. (A) Diffuse cytoplasmic expression in sarcomatoid component, composite score 3.1. (B) Diffuse membranous expression in carcinoma component, composite score 4.8.

Figure 4

Secreted protein acidic and rich in cysteine, 20× objective. (A) Diffuse cytoplasmic expression in sarcomatous component, composite score 4.8. (B) Diffuse cytoplasmic expression in carcinomatous component, composite score 3.0.

Figure 5

E-cadherin, 20× objective. (A) Negative expression in sarcomatous component, composite score 0.5. (B) Diffuse membranous expression in carcinomatous component, composite score 2.9.

Figure 6

N-cadherin, 20× objective. (A) Diffuse cytoplasmic expression in sarcomatous component, composite score 5.1. (B) Diffuse membranous expression in carcinomatous component, composite score 4.9.

Discussion

SRCC is a highly aggressive tumour composed of both epithelial and mesenchymal cells.2 3 The pathogenesis of the mesenchymal cells is not clearly understood, but it is assumed that SC undergoes a metaplastic process in which the tumour cells lose their epithelial characteristics and gain mesenchymal phenotypic qualities via EMT.2 4 It is the acquisition of the increased ability to migrate and metastasise that makes SRCC highly aggressive. The purpose of this study was to investigate whether SRCC is such an example of EMT.

In the present study, the average age at diagnosis was 59.8 years and the average tumour size was 10.0 cm. For RCC without SC, the average age was 63.1 years and average size was 6.0 cm for the years 1998–2002.21 Of the 21 SRCC cases, 23.8% were TNM stage 1, 9.5% were stage 2, 42.9% were stage 3 and 23.8% were stage 4. The majority of cases were stage 3 or 4 at diagnosis, with one case of distant metastasis to the lungs. In a study examining RCC without SC from 1993 to 2004, the majority of cases were stage 1 (50.6% were stage 1, 26.7% were stage 2 or 3, and 22.7% were stage 4).22 These data suggest that individuals with SRCC tend to be younger and present at more advanced stages, and the tumours are larger.

Snail is involved in inducing EMT in many carcinomas.5 7 Here, we demonstrated that Snail was more likely to be expressed in SC than CC of SRCC. However, 81% of CC also had high expression. While Snail expression was higher in SC, there was nevertheless a high level of expression in CC, supporting the notion that it pre-empts the initiation of EMT, before morphological mesenchymal phenotypic expression.

E- to N-cadherin switching is considered a hallmark of EMT. Loss of expression of E-cadherin and increased expression of N-cadherin has been demonstrated in the SC of many carcinomas.5 6 In breast carcinomas, N-cadherin has a dominant effect, enhancing tumour cell motility even when co-expressed with E-cadherin.18 This study demonstrated a high N-cadherin and low E-cadherin expression, as with other examples of EMT. In CC, we anticipated a high expression of E-cadherin and low expression of N-cadherin. In contrast, a high expression of both E- and N-cadherin in CC was found. Immunoexpression profiles suggest that high expression of N-cadherin within the tumour occurs prior to the mesenchymal phenotypic manifestation—in CC—emphasising the role in the initiation of EMT. Subsequently, the loss of E-cadherin corresponds to the SC seen in SRCC.

Interestingly, while there was high expression of N-cadherin in both SC and CC, the cellular localisation of N-cadherin was disparate. Cytoplasmic staining was seen in SC while membranous staining was seen in CC. This differential expression has been identified in pancreatic carcinomas17 and pleural mesotheliomas with sarcomatoid areas,23 and may reflect the difference in tumour cell adhesion between mesenchymal and epithelial components. The localisation of a cadherin to the membrane promotes cell–cell adhesion, while the movement of the cadherin from the membrane into the cytoplasm may promote cell motility.24 25 Hence this putative increased motility is likely associated with SRCC's aggressive behaviour.

E-cadherin is normally linked to the cytoskeleton by β-catenin. When cells undergo EMT, β-catenin dissociates from the membrane to the cytoplasm and then to the nucleus, where it acts as a transcriptional activator of Snail.5 7 16 In this study, we found that β-catenin was localised to the membrane in CC and to the cytoplasm in SC, as anticipated, but no nuclear expression was seen. This inexplicable event warrants further investigation.

Finally, SPARC is up-regulated in EMT. It plays a role in cell–extracellular matrix interaction by inducing disassembly of adhesion contacts or through activation of matrix-degrading enzymes, inducing motility in cells.20 In this study, it was more likely to be expressed in SC than CC, confirming that SC of SRCC have undergone EMT. Again, the presumed increased motility associated with SPARC is likely associated with the increased aggressive nature of SRCC.

The results of this study indicate that SRCC is an example of EMT. There is increased expression of Snail and SPARC, E- to N-cadherin switching, and dissociation of β-catenin from the membrane into the cytoplasm. The high expression of Snail and N-cadherin in CC suggests that they are involved in initiating EMT, prior to morphological mesenchymal phenotypic expression. Once EMT is established, the loss of E-cadherin, release of β-catenin into the cytoplasm, and expression of SPARC correspond with SC seen in SRCC. It is proposed that the acquisition of mesenchymal capability such as increased motility enables SRCC to present at advanced stages at diagnosis, implying a more aggressive behaviour.

Take-home messages

  • Sarcomatoid renal cell carcinoma (SRCC) is an example of epithelial–mesenchymal transition.

  • Using immunohistochemistry, the loss of expression of epithelial markers and a gain of mesenchymal markers has been shown in 21 cases of SRCC.

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References

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Footnotes

  • Competing interests None declared.

  • Ethics approval This study was conducted with the approval of the University of Vermont Institutional Review Board and Vermont Cancer Center Protocol Review Committee.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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