Advances in Medical Sciences 59 (2014) 142–146
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original Research Article
Gastric cancer patients less than 50 years of age exhibit significant downregulation of E-cadherin and CDX2 compared to older reference populations Claus Wilhelm Schildberg a,*,1, Mohammed Abba b,1, Susanne Merkel c, Abbas Agaimy d, Arno Dimmler e, Anne Schlabrakowski f, Roland Croner a, Jo¨rg Hendrik Leupold b, Werner Hohenberger a, Heike Allgayer b a
Department of Surgery, University Erlangen, Erlangen, Germany Department of Experimental Surgery and Molecular Oncology of Solid Tumors, Medical Faculty Mannheim, University Heidelberg and DKFZ Heidelberg, Heidelberg, Germany c Cancer Registry, Department of Surgery, University Erlangen, Erlangen, Germany d Institute of Pathology, University Erlangen, Erlangen, Germany e Department of Pathology, St. Vincentius Hospital, Karlsruhe, Germany f Department of Pathology, Klinikum Nord, Nu¨rnberg, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 October 2013 Accepted 12 November 2013 Available online 28 March 2014
Purpose: There is an increasing need to identify molecular markers, which can be used to prognosticate patient populations in gastric cancer. Whereas a significant number have been identified, very few have been characterized in the context of their ability to discriminate between young and old age groups in which a survival difference clearly exists. Material/methods: In this study, using immunohistochemistry, we evaluated three markers with proven involvement in gastric cancer. The p53 tumor suppressor, the cell adhesion glycoprotein epithelial cadherin (CDH1) and the caudal-related homeobox transcription factor (CDX2) all of these have important roles in the aetiopathogenesis and/or progression of gastric cancer. Results: After adjustments for TNM stage, tumor grade, histopathological characteristics (Lauren classification), we found significant differences in the expression of these proteins, particularly E-cadherin and CDX2 between young and elderly patients. However, these differences did not amount to a significant difference in survival. Conclusions: This study demonstrates that the protein expression of p53, CDH1 and CDX2 significantly discriminates young patients with gastric cancer who have a better prognostic outlook from older patients, but this difference in expression does not contribute to a survival benefit. ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Keywords: Gastric cancer Young patients Survival p53 CDX2
1. Introduction Worldwide, gastric cancer, including carcinomas of the gastroesophageal junction, constitutes the 4th and 5th most prevalent cancer in males and females, respectively [1]. According to recent presentations, this cancer type still accounts for an overall poor prognosis [1,2]. Several investigators have attempted to define risk
* Corresponding author at: Department of Surgery, University Erlangen, Krankenhausstrasse 12, 91054 Erlangen, Germany. Tel.: +49 9131 85 33296; fax: +49 9131 85 36596. E-mail address: [email protected] (C.W. Schildberg). 1 These authors contributed equally to this work.
categories that clearly delineate prognostic subgroups including tumor stage and chronological age amongst others with varying degrees of success [3–8]. Clinically, immunohistochemical markers are frequently being explored to support diagnosis and patient stratification, and in certain cases prognosticate especially amongst age groups [7,9–11]. Interestingly, relative to the number of molecules implicated in gastric carcinogenesis, those that have been explored as potential immunohistochemical markers with possible prognostic implications that discriminate between age groups are few. Very recently, we demonstrated that TFF1, Src and COX-2 could be explored in this regard and showed that young patients with gastric carcinoma had a significantly better age corrected 5-year survival rate compared to an older reference population [7]. However, further
http://dx.doi.org/10.1016/j.advms.2014.03.002 1896-1126/ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
C.W. Schildberg et al. / Advances in Medical Sciences 59 (2014) 142–146
molecules with equally significant implications in gastric cancer progression exist. For instance, several reports have demonstrated a strong involvement of E-cadherin, p53 and CDX2 in gastric cancer development, especially in advanced cancers [12–18]. TP53 is one of the most extensively studied tumor suppressor genes in human carcinogenesis. It is located on the short arm of chromosome 17, and encodes a 53 kDa protein that has established roles in cell cycle regulation, apoptosis, genome stabilization and angiogenesis [19,20]. The mutational continuum of p53 in gastric cancer is extensive. An increasing occurrence of p53 aberrations has been observed in the progression of gastric cancer, from gastritis all the way to advanced cases, with the highest frequency of abnormalities seen in metastatic lesions [21]. The caudal type homeobox 2 [CDX2] is a transcription factor belonging to the caudal-related homeobox gene family. It plays a role in determining trophoectoderm differentiation in embryogenesis; however, it also functions as a ‘master switch’ for intestinal differentiation, with expression in adults restricted to the small and large intestine [22]. CDX2 is also involved in the development and progression of gastric cancer [23,24]. Several studies have demonstrated that CDX2-positive expression in gastric cancer significantly correlates with better differentiation and a lower rate of lymph node metastasis [25–27]. The human E-cadherin gene encodes a calcium-dependent cellcell adhesion glycoprotein that is essential for development, cell differentiation and maintenance of epithelial polarity and structural integrity [28]. The suppression of E-cadherin is commonly observed in many sporadic tumors, and loss of function is thought to contribute to cancer progression by increasing proliferation, invasion, and metastasis. Around 25–40% of hereditary diffuse gastric cancers are caused by heterozygous E-cadherin. Reduced expression of E-cadherin correlates with an infiltrative and metastatic ability in gastric cancer [29]. In this study, we evaluated the protein expression of these three markers using immunohistochemistry and attempted to relate our findings to prognosis of patients older and younger than 50 years of age. 2. Material and methods 2.1. Patient information From 1st January 1995 to 31st December 2005, 423 chemotherapy naı¨ve gastric cancer cases encompassing carcinomas of the gastroesophageal junction (AEG II-III) were operated on at the surgical Department of the Erlangen University Hospital, Germany. Patients had a gastrectomy, transhiatal oesophagogastrectomy, or distal gastric resection. Adequate safety margins were taken into consideration with DII–III lymph node dissection, as well as reconstruction according to Billroth I, II or Roux Y. Data were collected and evaluated retrospectively. Patients who had preoperative or postoperative chemotherapy or radio-chemotherapy were excluded to enhance result comparisons and limit confounding variables, since this ensured that any deviations in the results would not be due to the effect of neoadjuvant or additive postoperative therapy. Out of the 423 patients, 56 were 50 years of age or younger at the time of diagnosis. The remaining 367 patients were over 50 years of age. Both groups were treated identically. The respective groups were matched on the basis of Lauren classifications, TNM staging (UICC 2009), and the R categories. The pair matching produced two groups of 40 patients each. The average follow-up period was 88 months (range 2–120 months).
circled, to match the blocks for the tissue microarray. Each target area on the selected block was punched to form a 0.6-mm diameter tissue core, and these were placed consecutively on recipient blocks of approximately 3 cm 2 cm, as previously described [30]. 2.3. Immunohistochemical analysis Immunohistochemistry (IHC) was performed on 3 mm sections using a polymer kit purchased from Zytomed Systems (Zytomed Systems Ltd., Berlin, Germany) with one of the following 3 antibodies according to the manufacturer’s instructions: E-cadherin (clone CX294, Dako, 1:30 dilution with pretreatment heating in TRS 9 buffer); p53 (clone 8G7G3/2, Zytomed, 1:3000 dilution with pretreatment heating in citric acid buffer) and CDX2 (polyclonal, DakoCytomation, Hamburg, Germany, 1:2500 dilution and no pretreatment). 2.4. Grading immunoreactivity For evaluating the results of the immunohistochemical stainings, the degree of immunoreactivity of the 3 antibodies (E-cadherin, p53 and CDX2) was quantitatively evaluated. For E-cadherin, a 4-grade scale was used: 0, absence of staining in tumor cells; 1+, weak nuclear and/or cytoplasmic staining in tumor cells; 2+, an intermediate staining intensity between 1+ and 3+ in tumor cells; and 3+, strong nuclear and cytoplasmic staining in tumor cells. For the other markers, a conventional percentage breakdown was used. Thus, a reference range that defines weak and strong expression is given for each marker. For E-cadherin, 0–1+ signifies weak expression, and all greater values indicate strong expression. For p53, 0–2% was defined as weak expression, while for CDX2, all values greater than 0–1% signified strong expression. To guarantee objectivity, two different pathologists carried out the assessment. 2.5. Statistical analysis The statistical analysis was performed using the SPSS statistical software for Windows (version 18.0, SPSS Inc., Chicago, Illinois, USA) and all survival data were analyzed for significance using the log rank test. Comparisons between frequencies were performed using the Chi-square test or, where appropriate, Fisher’s exact test. A p-value of
Contents lists available at ScienceDirect
Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original Research Article
Gastric cancer patients less than 50 years of age exhibit significant downregulation of E-cadherin and CDX2 compared to older reference populations Claus Wilhelm Schildberg a,*,1, Mohammed Abba b,1, Susanne Merkel c, Abbas Agaimy d, Arno Dimmler e, Anne Schlabrakowski f, Roland Croner a, Jo¨rg Hendrik Leupold b, Werner Hohenberger a, Heike Allgayer b a
Department of Surgery, University Erlangen, Erlangen, Germany Department of Experimental Surgery and Molecular Oncology of Solid Tumors, Medical Faculty Mannheim, University Heidelberg and DKFZ Heidelberg, Heidelberg, Germany c Cancer Registry, Department of Surgery, University Erlangen, Erlangen, Germany d Institute of Pathology, University Erlangen, Erlangen, Germany e Department of Pathology, St. Vincentius Hospital, Karlsruhe, Germany f Department of Pathology, Klinikum Nord, Nu¨rnberg, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 October 2013 Accepted 12 November 2013 Available online 28 March 2014
Purpose: There is an increasing need to identify molecular markers, which can be used to prognosticate patient populations in gastric cancer. Whereas a significant number have been identified, very few have been characterized in the context of their ability to discriminate between young and old age groups in which a survival difference clearly exists. Material/methods: In this study, using immunohistochemistry, we evaluated three markers with proven involvement in gastric cancer. The p53 tumor suppressor, the cell adhesion glycoprotein epithelial cadherin (CDH1) and the caudal-related homeobox transcription factor (CDX2) all of these have important roles in the aetiopathogenesis and/or progression of gastric cancer. Results: After adjustments for TNM stage, tumor grade, histopathological characteristics (Lauren classification), we found significant differences in the expression of these proteins, particularly E-cadherin and CDX2 between young and elderly patients. However, these differences did not amount to a significant difference in survival. Conclusions: This study demonstrates that the protein expression of p53, CDH1 and CDX2 significantly discriminates young patients with gastric cancer who have a better prognostic outlook from older patients, but this difference in expression does not contribute to a survival benefit. ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Keywords: Gastric cancer Young patients Survival p53 CDX2
1. Introduction Worldwide, gastric cancer, including carcinomas of the gastroesophageal junction, constitutes the 4th and 5th most prevalent cancer in males and females, respectively [1]. According to recent presentations, this cancer type still accounts for an overall poor prognosis [1,2]. Several investigators have attempted to define risk
* Corresponding author at: Department of Surgery, University Erlangen, Krankenhausstrasse 12, 91054 Erlangen, Germany. Tel.: +49 9131 85 33296; fax: +49 9131 85 36596. E-mail address: [email protected] (C.W. Schildberg). 1 These authors contributed equally to this work.
categories that clearly delineate prognostic subgroups including tumor stage and chronological age amongst others with varying degrees of success [3–8]. Clinically, immunohistochemical markers are frequently being explored to support diagnosis and patient stratification, and in certain cases prognosticate especially amongst age groups [7,9–11]. Interestingly, relative to the number of molecules implicated in gastric carcinogenesis, those that have been explored as potential immunohistochemical markers with possible prognostic implications that discriminate between age groups are few. Very recently, we demonstrated that TFF1, Src and COX-2 could be explored in this regard and showed that young patients with gastric carcinoma had a significantly better age corrected 5-year survival rate compared to an older reference population [7]. However, further
http://dx.doi.org/10.1016/j.advms.2014.03.002 1896-1126/ß 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
C.W. Schildberg et al. / Advances in Medical Sciences 59 (2014) 142–146
molecules with equally significant implications in gastric cancer progression exist. For instance, several reports have demonstrated a strong involvement of E-cadherin, p53 and CDX2 in gastric cancer development, especially in advanced cancers [12–18]. TP53 is one of the most extensively studied tumor suppressor genes in human carcinogenesis. It is located on the short arm of chromosome 17, and encodes a 53 kDa protein that has established roles in cell cycle regulation, apoptosis, genome stabilization and angiogenesis [19,20]. The mutational continuum of p53 in gastric cancer is extensive. An increasing occurrence of p53 aberrations has been observed in the progression of gastric cancer, from gastritis all the way to advanced cases, with the highest frequency of abnormalities seen in metastatic lesions [21]. The caudal type homeobox 2 [CDX2] is a transcription factor belonging to the caudal-related homeobox gene family. It plays a role in determining trophoectoderm differentiation in embryogenesis; however, it also functions as a ‘master switch’ for intestinal differentiation, with expression in adults restricted to the small and large intestine [22]. CDX2 is also involved in the development and progression of gastric cancer [23,24]. Several studies have demonstrated that CDX2-positive expression in gastric cancer significantly correlates with better differentiation and a lower rate of lymph node metastasis [25–27]. The human E-cadherin gene encodes a calcium-dependent cellcell adhesion glycoprotein that is essential for development, cell differentiation and maintenance of epithelial polarity and structural integrity [28]. The suppression of E-cadherin is commonly observed in many sporadic tumors, and loss of function is thought to contribute to cancer progression by increasing proliferation, invasion, and metastasis. Around 25–40% of hereditary diffuse gastric cancers are caused by heterozygous E-cadherin. Reduced expression of E-cadherin correlates with an infiltrative and metastatic ability in gastric cancer [29]. In this study, we evaluated the protein expression of these three markers using immunohistochemistry and attempted to relate our findings to prognosis of patients older and younger than 50 years of age. 2. Material and methods 2.1. Patient information From 1st January 1995 to 31st December 2005, 423 chemotherapy naı¨ve gastric cancer cases encompassing carcinomas of the gastroesophageal junction (AEG II-III) were operated on at the surgical Department of the Erlangen University Hospital, Germany. Patients had a gastrectomy, transhiatal oesophagogastrectomy, or distal gastric resection. Adequate safety margins were taken into consideration with DII–III lymph node dissection, as well as reconstruction according to Billroth I, II or Roux Y. Data were collected and evaluated retrospectively. Patients who had preoperative or postoperative chemotherapy or radio-chemotherapy were excluded to enhance result comparisons and limit confounding variables, since this ensured that any deviations in the results would not be due to the effect of neoadjuvant or additive postoperative therapy. Out of the 423 patients, 56 were 50 years of age or younger at the time of diagnosis. The remaining 367 patients were over 50 years of age. Both groups were treated identically. The respective groups were matched on the basis of Lauren classifications, TNM staging (UICC 2009), and the R categories. The pair matching produced two groups of 40 patients each. The average follow-up period was 88 months (range 2–120 months).
circled, to match the blocks for the tissue microarray. Each target area on the selected block was punched to form a 0.6-mm diameter tissue core, and these were placed consecutively on recipient blocks of approximately 3 cm 2 cm, as previously described [30]. 2.3. Immunohistochemical analysis Immunohistochemistry (IHC) was performed on 3 mm sections using a polymer kit purchased from Zytomed Systems (Zytomed Systems Ltd., Berlin, Germany) with one of the following 3 antibodies according to the manufacturer’s instructions: E-cadherin (clone CX294, Dako, 1:30 dilution with pretreatment heating in TRS 9 buffer); p53 (clone 8G7G3/2, Zytomed, 1:3000 dilution with pretreatment heating in citric acid buffer) and CDX2 (polyclonal, DakoCytomation, Hamburg, Germany, 1:2500 dilution and no pretreatment). 2.4. Grading immunoreactivity For evaluating the results of the immunohistochemical stainings, the degree of immunoreactivity of the 3 antibodies (E-cadherin, p53 and CDX2) was quantitatively evaluated. For E-cadherin, a 4-grade scale was used: 0, absence of staining in tumor cells; 1+, weak nuclear and/or cytoplasmic staining in tumor cells; 2+, an intermediate staining intensity between 1+ and 3+ in tumor cells; and 3+, strong nuclear and cytoplasmic staining in tumor cells. For the other markers, a conventional percentage breakdown was used. Thus, a reference range that defines weak and strong expression is given for each marker. For E-cadherin, 0–1+ signifies weak expression, and all greater values indicate strong expression. For p53, 0–2% was defined as weak expression, while for CDX2, all values greater than 0–1% signified strong expression. To guarantee objectivity, two different pathologists carried out the assessment. 2.5. Statistical analysis The statistical analysis was performed using the SPSS statistical software for Windows (version 18.0, SPSS Inc., Chicago, Illinois, USA) and all survival data were analyzed for significance using the log rank test. Comparisons between frequencies were performed using the Chi-square test or, where appropriate, Fisher’s exact test. A p-value of
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