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Expression of ICAM-1, E-cadherin, periostin and midkine in metastases of pancreatic ductal adenocarcinomas
Experimental and Molecular Pathology 104 (2018) 109–113
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Expression of ICAM-1, E-cadherin, periostin and midkine in metastases of pancreatic ductal adenocarcinomas
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Katharina Grupp , Nathaniel Melling, Valentina Bogoevska, Matthias Reeh, Faik Güntac Uzunoglu, Alexander Tarek El Gammal, Michael Fabian Nentwich, Jakob Robert Izbicki, Dean Bogoevski General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center Hamburg-Eppendorf, Martinistr. 52, Hamburg, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: ICAM-1 E-cadherin Periostin Midkine Pancreatic cancer Liver metastasis
Development and progression of malignant tumors is in part characterized by the ability of a tumor cell to overcome cell-cell and cell-matrix adhesion and to disseminate in organs distinct from that in which they originated. This study was undertaken to analyze the clinical significance of the expression of the following cell-cell and cell-matrix adhesion molecules in pancreatic ductal adenocarcinomas (PDACs) and synchronous liver metastases: intercellular adhesion molecule 1 (ICAM-1), E-cadherin, periostin, and midkine (MK). ICAM-1, Ecadherin, periostin and MK expression was analyzed by immunohistochemistry on a tissue microarray containing 34 PDACs and 12 liver metastasis specimens. ICAM-1 expression was predominantly localized in the membranes of the cells and was found in weak to moderate intensities in PDACs and liver metastases. E-cadherin expression was absent in the majority of PDACs and corresponding liver metastases. The secreted proteins periostin and MK were expressed in various intensities in primary cancers and liver metastases. Statistical analysis demonstrated that the expression levels of the analyzed markers were neither significantly associated with metastasis in PDACs nor with clinical outcome of patients. Our study shows that the expression of the cellcell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK was not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs.
1. Introduction Metastasis, the dissemination and growth of neoplastic cells in an organ distinct from that in which they originated (Fidler, 2002; Nguyen et al., 2009), is the most common cause of death in cancer patients. This is particularly true for pancreatic cancers, where most patients are diagnosed with metastatic disease (Stathis and Moore, 2010). In general, cancer invasion is a cell- and tissue-driven process for which the physical, cellular, and molecular determinants adapt and react throughout the progression of the disease (Friedl and Alexander, 2011). Cancer invasion is initiated and maintained by signaling pathways that control cytoskeletal dynamics in tumor cells and the turnover of cell-matrix and cell-cell junctions, followed by cell migration into the adjacent tissue (Friedl and Alexander, 2011). The best characterized alteration involves the loss of E-cadherin, a key cell-to-cell adhesion molecule in carcinoma cells (Hanahan and Weinberg, 2011). By
forming adherent junctions with adjacent epithelial cells, E-cadherin helps to assemble epithelial cell sheets and maintain the quiescence of the cells within these sheets (Hanahan and Weinberg, 2011). Increased expression of E-cadherin is well established as an antagonist of invasion and metastasis, whereas reduction of its expression is known to potentiate these phenotypes (Hanahan and Weinberg, 2011). Periostin, a component of the extracellular matrix is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumor (Malanchi et al., 2012). Infiltrating tumor cells need to induce stromal periostin expression in the secondary target organ to initiate colonization and blocking of the function of periostin prevents metastasis (Malanchi et al., 2012). ICAM-1 is a transmembrane molecule and a distinguished member of the immunoglobulin superfamily of proteins that participates in many important processes, including leukocyte endothelial transmigration, cell signaling, cell-cell interaction, cell polarity and tissue stability (Kotteas et al., 2014). ICAM-1 and its soluble part are
Abbreviations: PDACs, pancreatic ductal adenocarcinomas; ICAM-1, intercellular adhesion molecule 1; MK, midkine; PI3K, phosphatidylinositol 3-kinase ⁎ Corresponding author at: General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. E-mail addresses: [email protected] (K. Grupp), [email protected] (N. Melling), [email protected] (V. Bogoevska), [email protected] (F.G. Uzunoglu), [email protected] (A.T. El Gammal), [email protected] (M.F. Nentwich), [email protected] (J.R. Izbicki), [email protected] (D. Bogoevski). https://doi.org/10.1016/j.yexmp.2018.01.005 Received 24 September 2017; Accepted 10 January 2018 0014-4800/ © 2018 Published by Elsevier Inc.
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Primary antibodies specific for ICAM-1 (rabbit, New England Biolabs; at 1/100 dilution), E-cadherin (mouse, Dako; at 1/100 dilution), periostin (rabbit, Abcam; at 1/100 dilution) and MK (rabbit, Abcam; at 1/100 dilution) were applied, slides were deparaffinized and exposed to heatinduced antigen retrieval for 10 min in a microwave at 600 W. Bound antibody was then visualized using the AEC Chromogen. The immunostaining of ICAM-1, E-cadherin, periostin and MK was homogenous in the analyzed tissue samples and staining intensity of all cases was semiquantitatively assessed in four categories: negative, weak, moderate, and strong immunostaining.
highly expressed in inflammatory conditions, chronic diseases and a number of malignancies (Kotteas et al., 2014). MK belongs to the subfamily of heparin binding growth factors (Kadomatsu et al., 1988), prominently expressed during embryogenesis but down-regulated to negligible levels in healthy adults (Jones, 2014). The expression of MK is frequently upregulated in many types of human carcinoma and knockdown of MK suppresses tumorigenesis and tumor development (Sakamoto and Kadomatsu, 2012). Here, we analyze the clinical impact of the expression of the cell-cell and cell-matrix adhesion molecules E-cadherin, periostin, ICAM1, and MK in PDACs and liver metastasis by immunohistochemistry. Our study shows that the expression of the cell-cell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK was not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs.
2.3. Statistics Statistical calculations were performed with SPSS software (IBM Corporation, New York; USA). Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant survival differences between groups. COX proportional hazards regression analysis was performed to test the statistical independence and significance between pathological and clinical variables.
2. Materials and methods 2.1. Patients Primary pancreatic cancer specimens were available from 34 patients undergoing surgery between 1999 and 2009 at the Department of General, Visceral and Thoracic Surgery at the University Medical Center Hamburg-Eppendorf. 11 of these primary pancreatic cancers presented with synchronous metastases. The median follow-up time of the patients was 45 months (ranging between 3 and 180 months). Clinical and pathological characteristics of all patients on the TMA are summarized in Table 1. The TMA manufacturing process was described earlier in detail (Mirlacher et al. 2010). In short, one 0.6 mm core was taken from a representative tissue block from each patient. The tissues were distributed among the TMA containing all 34 primary pancreatic cancers with the 11 corresponding synchronous liver metastasis specimens. The local ethical committee of Hamburg approved this study. Informed consent was obtained from all patients before inclusion in the study.
3. Results 3.1. Technical issues A total of 2.9% of arrayed PDACs and 9.1% of liver metastases were non-informative for IHC due to the complete lack of tissue or absence of unequivocal cancer cells. 3.2. ICAM-1 ICAM-1 staining was predominantly localized in the cell membrane. ICAM-1 staining was positive in all interpretable PDACs and liver metastases and considered weak in 87.9% and 72.7% and moderate in 12.1% and 27.3% of cases. Representative pictures are shown in Fig. 1A.
2.2. Immunohistochemistry Freshly cut TMA sections were immunostained in one experiment.
3.3. E-cadherin
Table 1 Composition of the tissue microarray containing 34 PDCA specimens.
E-cadherin immunostaining was – if present – also predominantly localized in the cell membrane (Fig. 1B). E-cadherin staining was absent in 75% of analyzable PDACs and was considered weak in the other 25%. In liver metastases, E-cadherin staining was absent in all interpretable tissue samples.
No. of patients Study cohort on tissue microarray (n = 34) pT category pTis pT1 pT2 pT3 pT4
0 0 1 29 2
G category G1 G2 G3
3 13 15
pN category pN0 pN+
9 23
UICC category 0 1a 1b 2a 2b 3 4
0 0 0 7 13 1 11
3.4. Periostin Expression of the secreted protein periostin was found in various intensities in PDACs and corresponding liver metastases (Fig. 1C). In detail, Periostin expression was considered weak in 18.2% and 10%, moderate in 38.4% and 60%, and strong in 42.2% and 30% of interpretable PDACs and liver metastases. 3.5. MK MK expression was considered weak in 81.8% and 60% and moderate in 18.2% and 40% of PDAC and liver metastasis cases. Immunohistochemisty of MK is shown in Fig. 1D. 3.6. Clinical significance of the molecules Kaplan-Meier curves demonstrated that the expression of ICAM-1, E-cadherin, Periostin and MK was unrelated to clinical outcome of pancreatic cancer patients, as shown in Fig. 2.
Note: Numbers do not always add up to 34 in the different categories because of cases with missing data.
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Fig. 1. Representative pictures of (A) ICAM-1, (B) E-cadherin, (C) periostin and (D) midkine expression in primary pancreatic cancers and liver metastases.
4. Discussion
ICAM-1 expression is upregulated in malignant compared to normal pancreatic tissue suggesting that enhanced expression of ICAM-1 might contribute to cancer cell migration and the spread of cancer cells to distant organs (Tempia-Caliera et al., 2002; Shimoyama et al., 1997). Functional studies analyzing the influence of ICAM-1 on metastatic process in pancreatic cancer cells have been conflicting since both prometastatic and anti-metastatic roles of ICAM-1 have been described. Some studies suggested that increased expression of ICAM-1 in senescent human omentum-derived mesothelial cells facilitate peritoneal adhesion of selected pancreatic cancers (Ksiazek et al., 2010). In line with this study, interference with the function of ICAM-1 has been
The result of our study shows that the expression of the cell-cell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK was not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs. This is the first study analyzing ICAM-1 staining in PDACs and corresponding liver metastases. There were no statistical differences in the expression levels of ICAM-1 in the analyzed PDACs as compared to the corresponding liver metastases. Previous studies had suggested that
Fig. 2. Relationship of (A) ICAM-1, (B) E-cadherin, (C) periostin and (D) midkine expression with clinical outcome in primary pancreatic cancer patients.
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of cancer cells in vitro and in vivo (Katsuno et al., 2013). Additionally, MK is described to mediate cell survival and growth mainly through PI3K and extracellular signal-regulated kinase (ERK) signaling (Sandra et al 2004). Our study identified MK expression in various intensities in the majority of analyzed primary pancreatic cancers and liver metastasis. Despite small differences in the expression-intensities in the tissues, MK expression levels were unrelated to patient outcome in our TMA cohort. Earlier studies on MK expression in 75 pancreatic cancers described an expression of MK in 53.3% of interpretable cases linked to venous invasion, microvessel density, and liver metastasis (Maeda et al 2007). These divergent results may be due to differences in patient cohort or IHC protocols. For example, in our study an Abcam's MK antibody was used, while Maeda et al. (2007) applied an antibody from Santa Cruz Laboratory. In summary, our study shows that the expression of the cell-cell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK were not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs.
shown to reduce the ability of pancreatic cancer cell lines to adhere to the mesothelium (Ziprin et al., 2003), and has been suggested to decrease the incidence of peritoneal tumor recurrence after curative resection of pancreatic cancer (van Grevenstein et al., 2006). Other studies using ICAM-1-null (ICAM-1(−/−)) mice have shown that there was no significant difference in pancreatic cancer progression in wildtype versus ICAM-1 null mice (Roland et al., 2010) and have shown that anti-ICAM-1 monoclonal antibodies did not decrease adhesion potential of pancreatic cancer cells to microvascular endothelial cells (ten Kate et al., 2006). In our study, expression of ICAM-1 was unrelated to metastasis of pancreatic cancers and to prognosis of patients, suggesting that molecules other than ICAM-1 may contribute to metastatic potential of pancreatic cancer cells and may be useful as prognostic markers in PDACs. Next, we evaluated the clinical impact of E-cadherin expression in pancreatic cancer and liver metastasis. Physiologically, E-cadherin has been described to play an important role in cell-to-cell cohesion, cell-tocell recognition, and epithelial polarity (Jeanes et al., 2008). In cancer, E-cadherin has been described as a tumor suppressor molecule (Jeanes et al., 2008). The results of our study show that E-cadherin expression was absent in most of the analyzed tissue spots. This observation is in line with several previous studies describing a reduction/loss of Ecadherin in pancreatic cancer cells (Shimamura et al., 2003; Winter et al., 2008; Karayiannakis et al., 2001; Yonemasu et al., 2001; Li et al., 2003; Joo et al., 2002; Masugi et al., 2010; Oida et al., 2006; Shin et al., 2005; Pryczynicz et al., 2010; Hong et al., 2011). Recently, it has been purported that epithelial-mesenchymal transition (EMT) is crucial to cancer invasion and metastasis (Kalluri and Weinberg, 2009). The EMT phenotype is characterized by the loss of cell-to-cell adhesion with disintegration (Dorado et al., 2011) (Moore et al., 2007), a phenotypic change where cells shift from an” epithelial” morphology to an elongated fibroblast-like morphology which is associated with increased motility and tumor invasion (Hugo et al., 2007). The process of EMT involves the up-regulation of mesenchymal markers such as vimentin, N-cadherin and fibronectin, and the down-regulation of epithelial adhesion molecules such as E-cadherin and cytokeratins (Min et al., 2008; Larue and Bellacosa, 2005). EMT can be initiated by signaling pathways activated by tyrosine and serine-threonine kinase activity receptors (e.g. phosphatidylinositol 3-kinase (PI3K)) (Guarino, 2007), which in turn is regulated by periostin (Bao et al., 2004; Morra and Moch, 2011; Baril et al., 2007). Periostin was shown to be not only a marker of EMT, but to be itself an inducer of this phenomenon (Yan and Shao, 2006; Kim et al., 2011). Yan and Shao (2006) have demonstrated that ectopic expression of periostin in tumorigenic but non-metastatic 293T cells can induce EMT and promote invasion and metastasis in vivo. The upregulation of periostin expression was accompanied by the upregulation of vimentin, fibronectin, and active MMP-9, while the expression of Ecadherin and N-cadherin was unaltered (Yan and Shao, 2006). Periostin signaling pathway in 293T cells seems to require interaction with αvβ5 integrin and recruitment of EGFR (Yan and Shao, 2006). In turn, Kim et al. (2011) found that upregulation of Akt phosphorylation and Snail by periostin is involved in the regulation of E-cadherin and the invasiveness of prostate cancer cells. Besides the important role played by periostin in EMT, it is believed that this protein can also stimulate anigogenesis and lymphangiogenesis in tumors (Ratajczak-Wielgomas and Dziegiel, 2015). These data suggest that periostin may play an important role in the progression of cancer. In our study, periostin was expressed in various levels in all of the analyzable tissue samples suggesting an important role in pancreatic carcinogenesis. However, we observed no significant differences in expression intensities in primary PDACs and liver metastasis or in relation to survival of the patients. We performed the IHC analysis of MK, which has also been described to be linked to the EMT process (Katsuno et al., 2013). MK has been shown to interact with various protein members of the TGF-β pathway in vitro, a pathway that is well accepted to be a central mediator of EMT processes and consequently navigates increased migration
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Expression of ICAM-1, E-cadherin, periostin and midkine in metastases of pancreatic ductal adenocarcinomas
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Katharina Grupp , Nathaniel Melling, Valentina Bogoevska, Matthias Reeh, Faik Güntac Uzunoglu, Alexander Tarek El Gammal, Michael Fabian Nentwich, Jakob Robert Izbicki, Dean Bogoevski General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center Hamburg-Eppendorf, Martinistr. 52, Hamburg, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: ICAM-1 E-cadherin Periostin Midkine Pancreatic cancer Liver metastasis
Development and progression of malignant tumors is in part characterized by the ability of a tumor cell to overcome cell-cell and cell-matrix adhesion and to disseminate in organs distinct from that in which they originated. This study was undertaken to analyze the clinical significance of the expression of the following cell-cell and cell-matrix adhesion molecules in pancreatic ductal adenocarcinomas (PDACs) and synchronous liver metastases: intercellular adhesion molecule 1 (ICAM-1), E-cadherin, periostin, and midkine (MK). ICAM-1, Ecadherin, periostin and MK expression was analyzed by immunohistochemistry on a tissue microarray containing 34 PDACs and 12 liver metastasis specimens. ICAM-1 expression was predominantly localized in the membranes of the cells and was found in weak to moderate intensities in PDACs and liver metastases. E-cadherin expression was absent in the majority of PDACs and corresponding liver metastases. The secreted proteins periostin and MK were expressed in various intensities in primary cancers and liver metastases. Statistical analysis demonstrated that the expression levels of the analyzed markers were neither significantly associated with metastasis in PDACs nor with clinical outcome of patients. Our study shows that the expression of the cellcell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK was not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs.
1. Introduction Metastasis, the dissemination and growth of neoplastic cells in an organ distinct from that in which they originated (Fidler, 2002; Nguyen et al., 2009), is the most common cause of death in cancer patients. This is particularly true for pancreatic cancers, where most patients are diagnosed with metastatic disease (Stathis and Moore, 2010). In general, cancer invasion is a cell- and tissue-driven process for which the physical, cellular, and molecular determinants adapt and react throughout the progression of the disease (Friedl and Alexander, 2011). Cancer invasion is initiated and maintained by signaling pathways that control cytoskeletal dynamics in tumor cells and the turnover of cell-matrix and cell-cell junctions, followed by cell migration into the adjacent tissue (Friedl and Alexander, 2011). The best characterized alteration involves the loss of E-cadherin, a key cell-to-cell adhesion molecule in carcinoma cells (Hanahan and Weinberg, 2011). By
forming adherent junctions with adjacent epithelial cells, E-cadherin helps to assemble epithelial cell sheets and maintain the quiescence of the cells within these sheets (Hanahan and Weinberg, 2011). Increased expression of E-cadherin is well established as an antagonist of invasion and metastasis, whereas reduction of its expression is known to potentiate these phenotypes (Hanahan and Weinberg, 2011). Periostin, a component of the extracellular matrix is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumor (Malanchi et al., 2012). Infiltrating tumor cells need to induce stromal periostin expression in the secondary target organ to initiate colonization and blocking of the function of periostin prevents metastasis (Malanchi et al., 2012). ICAM-1 is a transmembrane molecule and a distinguished member of the immunoglobulin superfamily of proteins that participates in many important processes, including leukocyte endothelial transmigration, cell signaling, cell-cell interaction, cell polarity and tissue stability (Kotteas et al., 2014). ICAM-1 and its soluble part are
Abbreviations: PDACs, pancreatic ductal adenocarcinomas; ICAM-1, intercellular adhesion molecule 1; MK, midkine; PI3K, phosphatidylinositol 3-kinase ⁎ Corresponding author at: General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. E-mail addresses: [email protected] (K. Grupp), [email protected] (N. Melling), [email protected] (V. Bogoevska), [email protected] (F.G. Uzunoglu), [email protected] (A.T. El Gammal), [email protected] (M.F. Nentwich), [email protected] (J.R. Izbicki), [email protected] (D. Bogoevski). https://doi.org/10.1016/j.yexmp.2018.01.005 Received 24 September 2017; Accepted 10 January 2018 0014-4800/ © 2018 Published by Elsevier Inc.
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Primary antibodies specific for ICAM-1 (rabbit, New England Biolabs; at 1/100 dilution), E-cadherin (mouse, Dako; at 1/100 dilution), periostin (rabbit, Abcam; at 1/100 dilution) and MK (rabbit, Abcam; at 1/100 dilution) were applied, slides were deparaffinized and exposed to heatinduced antigen retrieval for 10 min in a microwave at 600 W. Bound antibody was then visualized using the AEC Chromogen. The immunostaining of ICAM-1, E-cadherin, periostin and MK was homogenous in the analyzed tissue samples and staining intensity of all cases was semiquantitatively assessed in four categories: negative, weak, moderate, and strong immunostaining.
highly expressed in inflammatory conditions, chronic diseases and a number of malignancies (Kotteas et al., 2014). MK belongs to the subfamily of heparin binding growth factors (Kadomatsu et al., 1988), prominently expressed during embryogenesis but down-regulated to negligible levels in healthy adults (Jones, 2014). The expression of MK is frequently upregulated in many types of human carcinoma and knockdown of MK suppresses tumorigenesis and tumor development (Sakamoto and Kadomatsu, 2012). Here, we analyze the clinical impact of the expression of the cell-cell and cell-matrix adhesion molecules E-cadherin, periostin, ICAM1, and MK in PDACs and liver metastasis by immunohistochemistry. Our study shows that the expression of the cell-cell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK was not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs.
2.3. Statistics Statistical calculations were performed with SPSS software (IBM Corporation, New York; USA). Survival curves were calculated according to Kaplan-Meier. The Log-Rank test was applied to detect significant survival differences between groups. COX proportional hazards regression analysis was performed to test the statistical independence and significance between pathological and clinical variables.
2. Materials and methods 2.1. Patients Primary pancreatic cancer specimens were available from 34 patients undergoing surgery between 1999 and 2009 at the Department of General, Visceral and Thoracic Surgery at the University Medical Center Hamburg-Eppendorf. 11 of these primary pancreatic cancers presented with synchronous metastases. The median follow-up time of the patients was 45 months (ranging between 3 and 180 months). Clinical and pathological characteristics of all patients on the TMA are summarized in Table 1. The TMA manufacturing process was described earlier in detail (Mirlacher et al. 2010). In short, one 0.6 mm core was taken from a representative tissue block from each patient. The tissues were distributed among the TMA containing all 34 primary pancreatic cancers with the 11 corresponding synchronous liver metastasis specimens. The local ethical committee of Hamburg approved this study. Informed consent was obtained from all patients before inclusion in the study.
3. Results 3.1. Technical issues A total of 2.9% of arrayed PDACs and 9.1% of liver metastases were non-informative for IHC due to the complete lack of tissue or absence of unequivocal cancer cells. 3.2. ICAM-1 ICAM-1 staining was predominantly localized in the cell membrane. ICAM-1 staining was positive in all interpretable PDACs and liver metastases and considered weak in 87.9% and 72.7% and moderate in 12.1% and 27.3% of cases. Representative pictures are shown in Fig. 1A.
2.2. Immunohistochemistry Freshly cut TMA sections were immunostained in one experiment.
3.3. E-cadherin
Table 1 Composition of the tissue microarray containing 34 PDCA specimens.
E-cadherin immunostaining was – if present – also predominantly localized in the cell membrane (Fig. 1B). E-cadherin staining was absent in 75% of analyzable PDACs and was considered weak in the other 25%. In liver metastases, E-cadherin staining was absent in all interpretable tissue samples.
No. of patients Study cohort on tissue microarray (n = 34) pT category pTis pT1 pT2 pT3 pT4
0 0 1 29 2
G category G1 G2 G3
3 13 15
pN category pN0 pN+
9 23
UICC category 0 1a 1b 2a 2b 3 4
0 0 0 7 13 1 11
3.4. Periostin Expression of the secreted protein periostin was found in various intensities in PDACs and corresponding liver metastases (Fig. 1C). In detail, Periostin expression was considered weak in 18.2% and 10%, moderate in 38.4% and 60%, and strong in 42.2% and 30% of interpretable PDACs and liver metastases. 3.5. MK MK expression was considered weak in 81.8% and 60% and moderate in 18.2% and 40% of PDAC and liver metastasis cases. Immunohistochemisty of MK is shown in Fig. 1D. 3.6. Clinical significance of the molecules Kaplan-Meier curves demonstrated that the expression of ICAM-1, E-cadherin, Periostin and MK was unrelated to clinical outcome of pancreatic cancer patients, as shown in Fig. 2.
Note: Numbers do not always add up to 34 in the different categories because of cases with missing data.
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Fig. 1. Representative pictures of (A) ICAM-1, (B) E-cadherin, (C) periostin and (D) midkine expression in primary pancreatic cancers and liver metastases.
4. Discussion
ICAM-1 expression is upregulated in malignant compared to normal pancreatic tissue suggesting that enhanced expression of ICAM-1 might contribute to cancer cell migration and the spread of cancer cells to distant organs (Tempia-Caliera et al., 2002; Shimoyama et al., 1997). Functional studies analyzing the influence of ICAM-1 on metastatic process in pancreatic cancer cells have been conflicting since both prometastatic and anti-metastatic roles of ICAM-1 have been described. Some studies suggested that increased expression of ICAM-1 in senescent human omentum-derived mesothelial cells facilitate peritoneal adhesion of selected pancreatic cancers (Ksiazek et al., 2010). In line with this study, interference with the function of ICAM-1 has been
The result of our study shows that the expression of the cell-cell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK was not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs. This is the first study analyzing ICAM-1 staining in PDACs and corresponding liver metastases. There were no statistical differences in the expression levels of ICAM-1 in the analyzed PDACs as compared to the corresponding liver metastases. Previous studies had suggested that
Fig. 2. Relationship of (A) ICAM-1, (B) E-cadherin, (C) periostin and (D) midkine expression with clinical outcome in primary pancreatic cancer patients.
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of cancer cells in vitro and in vivo (Katsuno et al., 2013). Additionally, MK is described to mediate cell survival and growth mainly through PI3K and extracellular signal-regulated kinase (ERK) signaling (Sandra et al 2004). Our study identified MK expression in various intensities in the majority of analyzed primary pancreatic cancers and liver metastasis. Despite small differences in the expression-intensities in the tissues, MK expression levels were unrelated to patient outcome in our TMA cohort. Earlier studies on MK expression in 75 pancreatic cancers described an expression of MK in 53.3% of interpretable cases linked to venous invasion, microvessel density, and liver metastasis (Maeda et al 2007). These divergent results may be due to differences in patient cohort or IHC protocols. For example, in our study an Abcam's MK antibody was used, while Maeda et al. (2007) applied an antibody from Santa Cruz Laboratory. In summary, our study shows that the expression of the cell-cell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK were not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs.
shown to reduce the ability of pancreatic cancer cell lines to adhere to the mesothelium (Ziprin et al., 2003), and has been suggested to decrease the incidence of peritoneal tumor recurrence after curative resection of pancreatic cancer (van Grevenstein et al., 2006). Other studies using ICAM-1-null (ICAM-1(−/−)) mice have shown that there was no significant difference in pancreatic cancer progression in wildtype versus ICAM-1 null mice (Roland et al., 2010) and have shown that anti-ICAM-1 monoclonal antibodies did not decrease adhesion potential of pancreatic cancer cells to microvascular endothelial cells (ten Kate et al., 2006). In our study, expression of ICAM-1 was unrelated to metastasis of pancreatic cancers and to prognosis of patients, suggesting that molecules other than ICAM-1 may contribute to metastatic potential of pancreatic cancer cells and may be useful as prognostic markers in PDACs. Next, we evaluated the clinical impact of E-cadherin expression in pancreatic cancer and liver metastasis. Physiologically, E-cadherin has been described to play an important role in cell-to-cell cohesion, cell-tocell recognition, and epithelial polarity (Jeanes et al., 2008). In cancer, E-cadherin has been described as a tumor suppressor molecule (Jeanes et al., 2008). The results of our study show that E-cadherin expression was absent in most of the analyzed tissue spots. This observation is in line with several previous studies describing a reduction/loss of Ecadherin in pancreatic cancer cells (Shimamura et al., 2003; Winter et al., 2008; Karayiannakis et al., 2001; Yonemasu et al., 2001; Li et al., 2003; Joo et al., 2002; Masugi et al., 2010; Oida et al., 2006; Shin et al., 2005; Pryczynicz et al., 2010; Hong et al., 2011). Recently, it has been purported that epithelial-mesenchymal transition (EMT) is crucial to cancer invasion and metastasis (Kalluri and Weinberg, 2009). The EMT phenotype is characterized by the loss of cell-to-cell adhesion with disintegration (Dorado et al., 2011) (Moore et al., 2007), a phenotypic change where cells shift from an” epithelial” morphology to an elongated fibroblast-like morphology which is associated with increased motility and tumor invasion (Hugo et al., 2007). The process of EMT involves the up-regulation of mesenchymal markers such as vimentin, N-cadherin and fibronectin, and the down-regulation of epithelial adhesion molecules such as E-cadherin and cytokeratins (Min et al., 2008; Larue and Bellacosa, 2005). EMT can be initiated by signaling pathways activated by tyrosine and serine-threonine kinase activity receptors (e.g. phosphatidylinositol 3-kinase (PI3K)) (Guarino, 2007), which in turn is regulated by periostin (Bao et al., 2004; Morra and Moch, 2011; Baril et al., 2007). Periostin was shown to be not only a marker of EMT, but to be itself an inducer of this phenomenon (Yan and Shao, 2006; Kim et al., 2011). Yan and Shao (2006) have demonstrated that ectopic expression of periostin in tumorigenic but non-metastatic 293T cells can induce EMT and promote invasion and metastasis in vivo. The upregulation of periostin expression was accompanied by the upregulation of vimentin, fibronectin, and active MMP-9, while the expression of Ecadherin and N-cadherin was unaltered (Yan and Shao, 2006). Periostin signaling pathway in 293T cells seems to require interaction with αvβ5 integrin and recruitment of EGFR (Yan and Shao, 2006). In turn, Kim et al. (2011) found that upregulation of Akt phosphorylation and Snail by periostin is involved in the regulation of E-cadherin and the invasiveness of prostate cancer cells. Besides the important role played by periostin in EMT, it is believed that this protein can also stimulate anigogenesis and lymphangiogenesis in tumors (Ratajczak-Wielgomas and Dziegiel, 2015). These data suggest that periostin may play an important role in the progression of cancer. In our study, periostin was expressed in various levels in all of the analyzable tissue samples suggesting an important role in pancreatic carcinogenesis. However, we observed no significant differences in expression intensities in primary PDACs and liver metastasis or in relation to survival of the patients. We performed the IHC analysis of MK, which has also been described to be linked to the EMT process (Katsuno et al., 2013). MK has been shown to interact with various protein members of the TGF-β pathway in vitro, a pathway that is well accepted to be a central mediator of EMT processes and consequently navigates increased migration
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