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Epithelial-to-mesenchymal transition in endometrioid adenocarcinoma of the endometrium
Human Pathology (2013) 44, 1956–1963
www.elsevier.com/locate/humpath
Correspondence
Epithelial-to-mesenchymal transition in endometrioid adenocarcinoma of the endometrium To the Editor, I read with interest the recent article by Montserrat et al [1] describing epithelial-to-mesenchymal transition (EMT) in stage I endometrioid adenocarcinoma of the endometrium (EEC). The authors demonstrated reduced expression of E-cadherin and increased expression of the E-cadherin repressors Zinc finger protein SNAI1 (SNAIL), Zinc finger protein SNAI2 (SLUG), Zinc finger E-box-binding homeobox 1 (ZEB1), High mobility group protein HMGI-C (HMGA2), and Twist-related protein (TWIST) in tumor tissue compared with normal endometrium. There was increased SLUG, ZEB1, and HMGA2 messenger RNA expression in stage IC EEC compared with nonmyoinvasive tumors, and similar differences were present at the invasive front of more deeply invasive carcinomas compared with corresponding superficial tumor samples. The authors also showed an inverse correlation between E-cadherin expression and SNAIL and Twist protein expression. Finally, the authors induced EMT-like changes in the Ishikawa EEC cell line by infection with a lentivirus harboring the V600 BRAF mutation. The EMT phenotypic changes could be abrogated by MEK1/2 inhibition [1]. This interesting study lends further support to the hypothesis that EMT plays a role in EEC progression and, in particular, contributes toward the process of myometrial invasion. In earlier studies, we demonstrated immunophenotypic changes that were restricted mainly to the invasive front of EEC, including loss of E-cadherin that we postulated might represent EMT [2]. Furthermore, these changes often were accompanied by a localized increase in cyclin D1, p16 protein, and fascin expression and more consistent cytokeratin 7 and 19 immunoreactivity but loss of hormone receptor staining in the tumor cells [2-5]. In contrast to the study of Montserrat et al, which mainly used tissue microarray preparations, we used standard full face sections and found that the aforementioned changes were usually restricted to tumor areas showing the “MELF” (microcystic, elongated, and fragmented) type of morphological features. There was not a “global” loss of E-cadherin expression at the invasive front of the deeply myoinvasive tumor components. The authors do not comment 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
whether the immunophenotypic changes that they describe at the invasive tumor front were associated with morphological changes in the neoplastic epithelium (as might be expected in EMT), although this does not seem to be the case in the illustrations provided (Figure 3, see [1]). It would also be interesting to know whether there was an association between protein expression and changes in the surrounding stroma because it is known that reactive stromal factors play an important role in driving EMT processes. The in vitro component of the current study investigated mitogen-activated protein kinase (MAPK) pathway activation, which is known to induce EMT. As the authors note, an activating BRAF mutation such as that used in their study appears to be relatively rare in EEC, but this is not the case for KRAS mutations, which are present in 10% to 30% of cases [6]. Previously, we showed that EEC exhibiting EMT/ MELF changes more frequently showed KRAS mutations than tumors lacking MELF (45% versus 30%), although the differences did not reach statistical significance [7]. Nevertheless, there is increasing evidence based upon studies like that of Montserrat et al [1] to suggest that EMT may have a significant role in the EEC invasion, and this is likely to be of increasing relevance therapeutically in view of recent data suggesting a correlation between EMT and cancer stem cell characteristics [8]. Colin J.R. Stewart FRCPA King Edward Memorial Hospital, Histopathology Bagot Road, Subiaco, Perth, Western Australia 6008 Australia E-mail address: [email protected] http://dx.doi.org/10.1016/j.humpath.2013.04.020
References [1] Montserrat N, Mozos A, Llobet D, et al. Epithelial to mesenchymal transition in early stage endometrioid endometrial carcinoma. HUM PATHOL 2012;43:632-43. [2] Stewart CJR, Little L. Immunophenotypic features of MELF pattern invasion in endometrial adenocarcinoma: evidence for epithelialmesenchymal transition. Histopathology 2009;55:91-101. [3] Stewart CJR, Crook ML, Leung YC, Platten M. Expression of cell cycle regulatory proteins in endometrial adenocarcinoma: variations in
Correspondence
[4]
[5]
[6]
[7]
[8]
conventional tumor areas and in microcystic, elongated and fragmented glands. Mod Pathol 2009;22:725-33. Stewart CJR, Crook ML, Lacey J, Louwen K. Cytokeratin 19 expression in normal endometrium and in low-grade endometrioid adenocarcinoma of the endometrium. Int J Gynecol Pathol 2011;30:484-91. Stewart CJR, Crook ML, Manso L. Fascin expression in low-grade uterine endometrioid adenocarcinoma: correlation with microcystic, elongated and fragmented (MELF)–type alteration at the deep invasive margin. Histopathology 2011;59:73-80. Llobet D, Pallares J, Yeramian A, et al. Molecular pathology of endometrial carcinoma: practical aspects from the diagnostic and therapeutical viewpoints. J Clin Pathol 2009;62:777-85. Stewart CJR, Amanuel B, Grieu F, Carello A, Iacopetta B. KRAS mutation and microsatellite instability in endometrial adenocarcinomas showing MELF-type myometrial invasion. J Clin Pathol 2010;63: 604-8. Biddle A, Mackenzie IC. Cancer stem cells and EMT in carcinoma. Cancer Metastasis Rev 2012 http://dx.doi.org/10.100/s105555-012-9345-0.
1957 [3]. Moreover, it is also known that tumor-associated macrophages as well as tumor-associated fibroblasts produce hypoxia-inducible factor 1α, a potent inducer of EMT [4]. Likewise, nuclear factor κB may also directly activate the expression of EMT inducers, such as SNAIL and ZEB1 [5]. Although we have not directly related EMT with microenvironment, as we believe it was beyond the scope of our article, all these data suggest that the different stromal responses that we observe in EEC may also be related to EMT. Ana Mozos MD, PhD Jaime Prat MD, FRCP Hospital de la Santa Creu i Sant Pau, Department of Pathology Sant Antoni M. Claret, 167, Barcelona, 08025, Spain E-mail address: [email protected] http://dx.doi.org/10.1016/j.humpath.2013.04.023
Epithelial-to-mesenchymal transition in endometrioid adenocarcinoma of the endometrium—reply To the Editor: We thank Dr Stewart for his interest in our article [1]. As he points out, the expression of epithelial-to-mesenchymal transition (EMT) markers, such as E-cadherin, SNAIL, SLUG, TWIST, ZEB1, HMGA2, Mitogen-activated protein kinase 3 and 1 (ERK1/2), and p-ERK1/2 might be quite heterogeneous and, therefore, sometimes difficult to assess. As we acknowledged this fact, we evaluated in a subset of cases the expression of these EMT markers in whole tissue sections (unpublished data). We found that, in our series, the expression of most markers was homogeneous except for ERK1/2 and p-ERK1/2, as shown in Figures 3 and 4 [1]. Therefore, we studied E-cadherin, SNAIL, SLUG, TWIST, ZEB1, and HMGA2 using a tissue microarray approach, whereas we preferred full sections for ERK1/2 and p-ERK1/ 2 immunohistochemical studies. Moreover, as it is shown in Figures 3 and 4 [1], the expression of EMT markers was seen in areas of conventional endometrial endometrioid carcinoma (EEC) without microcystic, elongated, and fragmented morphological features. In our group, we have recently studied the expression of 2 distinct stromal signatures in a large series of EEC, namely, desmoid-type fibromatosis (DTF)-like or fibroblast-predominant response and colony stimulating factor 1 (CSF1) or macrophage-type response, as previously described [2]. We found that the different stromal signatures correlated with clinicopathologic features in EEC (unpublished data, manuscript in preparation), as may be observed in other cancer types. In our series, cases with the macrophage (CSF1) signature were of higher tumor grade and showed vascular invasion and phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), catalytic subunit α, mutations. It is known that the macrophage signature is induced by the CSF1 produced by tumor cells through the activation of the nuclear factor κB pathway, frequently activated in EEC
References [1] Montserrat N, Mozos A, Llobet D, et al. Epithelial to mesenchymal transition in early stage endometrioid endometrial carcinoma. HUM PATHOL 2012;43:632-43. [2] Sharma M, Beck AH, Webster JA, et al. Analysis of stromal signatures in the tumor microenvironment of ductal carcinoma in situ. Breast Cancer Res Treat 2010;123:397-404. [3] Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 2005;5: 749-59. [4] Cheng ZX, Sun B, Wang SJ, et al. Nuclear factor-kappaB-dependent epithelial to mesenchymal transition induced by HIF-1alpha activation in pancreatic cancer cells under hypoxic conditions. PLoS One 2011;6: e23752. [5] Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, Nakshatri H. NF-kappaB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene 2007;26:711-24.
Claudin-7 expression and its association with tumor progression in systemic malignancies To the Editor, Lappi-Blanco et al [1] have provided interesting data in their recent article. Claudin-7 expression may play a major role in tumor progression in a number of systemic malignancies. For instance, claudin-7 plays a role in the pathogenesis and progression of esophageal malignancies. Claudin-7 in esophageal carcinomas is mostly cytoplasmic. Typically, esophageal carcinomas demonstrate decreased claudin-7 expression [2]. Decreased claudin-7 expression, in turn, leads to attenuation of E-cadherin expression. This, in turn, leads to increased tumor invasiveness. Similarly, increased metastasis is seen in tumors that exhibit more down-regulated claudin-7 levels. In fact, a close association exists between claudin-7 expression and tumor stage [3]. Similarly, intestinal-type gastric carcinomas
www.elsevier.com/locate/humpath
Correspondence
Epithelial-to-mesenchymal transition in endometrioid adenocarcinoma of the endometrium To the Editor, I read with interest the recent article by Montserrat et al [1] describing epithelial-to-mesenchymal transition (EMT) in stage I endometrioid adenocarcinoma of the endometrium (EEC). The authors demonstrated reduced expression of E-cadherin and increased expression of the E-cadherin repressors Zinc finger protein SNAI1 (SNAIL), Zinc finger protein SNAI2 (SLUG), Zinc finger E-box-binding homeobox 1 (ZEB1), High mobility group protein HMGI-C (HMGA2), and Twist-related protein (TWIST) in tumor tissue compared with normal endometrium. There was increased SLUG, ZEB1, and HMGA2 messenger RNA expression in stage IC EEC compared with nonmyoinvasive tumors, and similar differences were present at the invasive front of more deeply invasive carcinomas compared with corresponding superficial tumor samples. The authors also showed an inverse correlation between E-cadherin expression and SNAIL and Twist protein expression. Finally, the authors induced EMT-like changes in the Ishikawa EEC cell line by infection with a lentivirus harboring the V600 BRAF mutation. The EMT phenotypic changes could be abrogated by MEK1/2 inhibition [1]. This interesting study lends further support to the hypothesis that EMT plays a role in EEC progression and, in particular, contributes toward the process of myometrial invasion. In earlier studies, we demonstrated immunophenotypic changes that were restricted mainly to the invasive front of EEC, including loss of E-cadherin that we postulated might represent EMT [2]. Furthermore, these changes often were accompanied by a localized increase in cyclin D1, p16 protein, and fascin expression and more consistent cytokeratin 7 and 19 immunoreactivity but loss of hormone receptor staining in the tumor cells [2-5]. In contrast to the study of Montserrat et al, which mainly used tissue microarray preparations, we used standard full face sections and found that the aforementioned changes were usually restricted to tumor areas showing the “MELF” (microcystic, elongated, and fragmented) type of morphological features. There was not a “global” loss of E-cadherin expression at the invasive front of the deeply myoinvasive tumor components. The authors do not comment 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
whether the immunophenotypic changes that they describe at the invasive tumor front were associated with morphological changes in the neoplastic epithelium (as might be expected in EMT), although this does not seem to be the case in the illustrations provided (Figure 3, see [1]). It would also be interesting to know whether there was an association between protein expression and changes in the surrounding stroma because it is known that reactive stromal factors play an important role in driving EMT processes. The in vitro component of the current study investigated mitogen-activated protein kinase (MAPK) pathway activation, which is known to induce EMT. As the authors note, an activating BRAF mutation such as that used in their study appears to be relatively rare in EEC, but this is not the case for KRAS mutations, which are present in 10% to 30% of cases [6]. Previously, we showed that EEC exhibiting EMT/ MELF changes more frequently showed KRAS mutations than tumors lacking MELF (45% versus 30%), although the differences did not reach statistical significance [7]. Nevertheless, there is increasing evidence based upon studies like that of Montserrat et al [1] to suggest that EMT may have a significant role in the EEC invasion, and this is likely to be of increasing relevance therapeutically in view of recent data suggesting a correlation between EMT and cancer stem cell characteristics [8]. Colin J.R. Stewart FRCPA King Edward Memorial Hospital, Histopathology Bagot Road, Subiaco, Perth, Western Australia 6008 Australia E-mail address: [email protected] http://dx.doi.org/10.1016/j.humpath.2013.04.020
References [1] Montserrat N, Mozos A, Llobet D, et al. Epithelial to mesenchymal transition in early stage endometrioid endometrial carcinoma. HUM PATHOL 2012;43:632-43. [2] Stewart CJR, Little L. Immunophenotypic features of MELF pattern invasion in endometrial adenocarcinoma: evidence for epithelialmesenchymal transition. Histopathology 2009;55:91-101. [3] Stewart CJR, Crook ML, Leung YC, Platten M. Expression of cell cycle regulatory proteins in endometrial adenocarcinoma: variations in
Correspondence
[4]
[5]
[6]
[7]
[8]
conventional tumor areas and in microcystic, elongated and fragmented glands. Mod Pathol 2009;22:725-33. Stewart CJR, Crook ML, Lacey J, Louwen K. Cytokeratin 19 expression in normal endometrium and in low-grade endometrioid adenocarcinoma of the endometrium. Int J Gynecol Pathol 2011;30:484-91. Stewart CJR, Crook ML, Manso L. Fascin expression in low-grade uterine endometrioid adenocarcinoma: correlation with microcystic, elongated and fragmented (MELF)–type alteration at the deep invasive margin. Histopathology 2011;59:73-80. Llobet D, Pallares J, Yeramian A, et al. Molecular pathology of endometrial carcinoma: practical aspects from the diagnostic and therapeutical viewpoints. J Clin Pathol 2009;62:777-85. Stewart CJR, Amanuel B, Grieu F, Carello A, Iacopetta B. KRAS mutation and microsatellite instability in endometrial adenocarcinomas showing MELF-type myometrial invasion. J Clin Pathol 2010;63: 604-8. Biddle A, Mackenzie IC. Cancer stem cells and EMT in carcinoma. Cancer Metastasis Rev 2012 http://dx.doi.org/10.100/s105555-012-9345-0.
1957 [3]. Moreover, it is also known that tumor-associated macrophages as well as tumor-associated fibroblasts produce hypoxia-inducible factor 1α, a potent inducer of EMT [4]. Likewise, nuclear factor κB may also directly activate the expression of EMT inducers, such as SNAIL and ZEB1 [5]. Although we have not directly related EMT with microenvironment, as we believe it was beyond the scope of our article, all these data suggest that the different stromal responses that we observe in EEC may also be related to EMT. Ana Mozos MD, PhD Jaime Prat MD, FRCP Hospital de la Santa Creu i Sant Pau, Department of Pathology Sant Antoni M. Claret, 167, Barcelona, 08025, Spain E-mail address: [email protected] http://dx.doi.org/10.1016/j.humpath.2013.04.023
Epithelial-to-mesenchymal transition in endometrioid adenocarcinoma of the endometrium—reply To the Editor: We thank Dr Stewart for his interest in our article [1]. As he points out, the expression of epithelial-to-mesenchymal transition (EMT) markers, such as E-cadherin, SNAIL, SLUG, TWIST, ZEB1, HMGA2, Mitogen-activated protein kinase 3 and 1 (ERK1/2), and p-ERK1/2 might be quite heterogeneous and, therefore, sometimes difficult to assess. As we acknowledged this fact, we evaluated in a subset of cases the expression of these EMT markers in whole tissue sections (unpublished data). We found that, in our series, the expression of most markers was homogeneous except for ERK1/2 and p-ERK1/2, as shown in Figures 3 and 4 [1]. Therefore, we studied E-cadherin, SNAIL, SLUG, TWIST, ZEB1, and HMGA2 using a tissue microarray approach, whereas we preferred full sections for ERK1/2 and p-ERK1/ 2 immunohistochemical studies. Moreover, as it is shown in Figures 3 and 4 [1], the expression of EMT markers was seen in areas of conventional endometrial endometrioid carcinoma (EEC) without microcystic, elongated, and fragmented morphological features. In our group, we have recently studied the expression of 2 distinct stromal signatures in a large series of EEC, namely, desmoid-type fibromatosis (DTF)-like or fibroblast-predominant response and colony stimulating factor 1 (CSF1) or macrophage-type response, as previously described [2]. We found that the different stromal signatures correlated with clinicopathologic features in EEC (unpublished data, manuscript in preparation), as may be observed in other cancer types. In our series, cases with the macrophage (CSF1) signature were of higher tumor grade and showed vascular invasion and phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), catalytic subunit α, mutations. It is known that the macrophage signature is induced by the CSF1 produced by tumor cells through the activation of the nuclear factor κB pathway, frequently activated in EEC
References [1] Montserrat N, Mozos A, Llobet D, et al. Epithelial to mesenchymal transition in early stage endometrioid endometrial carcinoma. HUM PATHOL 2012;43:632-43. [2] Sharma M, Beck AH, Webster JA, et al. Analysis of stromal signatures in the tumor microenvironment of ductal carcinoma in situ. Breast Cancer Res Treat 2010;123:397-404. [3] Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 2005;5: 749-59. [4] Cheng ZX, Sun B, Wang SJ, et al. Nuclear factor-kappaB-dependent epithelial to mesenchymal transition induced by HIF-1alpha activation in pancreatic cancer cells under hypoxic conditions. PLoS One 2011;6: e23752. [5] Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, Nakshatri H. NF-kappaB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene 2007;26:711-24.
Claudin-7 expression and its association with tumor progression in systemic malignancies To the Editor, Lappi-Blanco et al [1] have provided interesting data in their recent article. Claudin-7 expression may play a major role in tumor progression in a number of systemic malignancies. For instance, claudin-7 plays a role in the pathogenesis and progression of esophageal malignancies. Claudin-7 in esophageal carcinomas is mostly cytoplasmic. Typically, esophageal carcinomas demonstrate decreased claudin-7 expression [2]. Decreased claudin-7 expression, in turn, leads to attenuation of E-cadherin expression. This, in turn, leads to increased tumor invasiveness. Similarly, increased metastasis is seen in tumors that exhibit more down-regulated claudin-7 levels. In fact, a close association exists between claudin-7 expression and tumor stage [3]. Similarly, intestinal-type gastric carcinomas