{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:50593"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:50593","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Identifizierung und Charakterisierung von neuen DNA-Methylierungs-Biomarkern für das humane Mammakarzinom","abstract":"Molecular biomarkers, being capable of detecting the earliest forms of a malignancy or predicting the course of a cancer disease are of constantly growing interest in modern medicine. Such biomarkers will be useful in early cancer diagnosis, as well as in the prediction of a tumor’s response to a treatment regimen, and in combination with improved prognostication may help the clinician to select the most appropriate treatment option for the benefit of a cancer patient. Since aberrant DNA methylation is essentially involved in tumorigenesis, this alteration may provide a rich source of such molecular biomarkers. In this study, six genes were analyzed for a potential utility as a DNA methylation biomarker in human breast cancer. Five of these genes (SFRP1, SFRP2, SFRP5, WIF1 und DKK3) encode secreted inhibitors of the Wnt signaling pathway, which regulates fundamental processes, such as cell proliferation and differentiation. ITIH5 encodes an extracellular matrix protein, being involved in extracellular matrix integrity and in cell adhesion. As a prerequisite, each of the selected genes contains a CpG island within the gene promoter, being a potential substrate to aberrant DNA methylation. Initially, benign and malignant breast cell lines were selected for mRNA expression analysis of the respective genes by RT-PCR/realtime PCR, and also for promoter methylation analysis by methylation-specific PCR (MSP) after genomic DNA bisulfite conversion. Malignant cell lines commonly showed increased methylation in the analyzed gene promoters in close association with downregulation of mRNA transcripts, whereas unmethylated genes exhibited abundant mRNA expression. This association was functionally confirmed, since reduced mRNA expression of each methylated gene could be significantly induced in vitro by a DNA demethylating treatment with 5-aza-2’-deoxycytidine and trichostatin A. Therefore, promoter methylation was identified as the major molecular aberration leading to a common expression loss of the analyzed genes in malignant breast cell lines. Next, promoter methylation was investigated in a large cohort of primary breast carcinomas (n=204) and corresponding normal breast tissues (n=26) by MSP. This revealed a high frequency of promoter methylation in breast carcinomas (SFRP1: 81%; SFRP2: 83%; SFRP5: 73%; WIF1: 62%; DKK3: 61%; ITIH5: 68%), whereas in normal breast tissues these genes were almost completely unmethylated. Therefore, promoter methylation of the analyzed genes represents a tumor-specific alteration in human breast cancer (P<0.0001). For SFRP1, SFRP5, DKK3 and ITIH5, mRNA expression was determined in parallel to MSP for selected samples by realtime PCR. A detected frequent loss of mRNA expression (SFRP1: 87%; SFRP5: 62%; DKK3: 70%, ITIH5: 77%) was significantly associated with promoter methylation of the respective gene. Furthermore, SFRP2, DKK3 and ITIH5 expression was also quantified at the protein level by immunohistochemical staining, and revealed a frequent expression loss of SFRP2 in 75%, DKK3 in 74% and ITIH5 in 70% of the investigated breast carcinomas. Collectively, a high concordance between the frequency of promoter methylation and the frequency of expression loss could be detected for each analyzed gene. Taken together with the results from breast cell lines, promoter methylation represents the predominant molecular aberration that leads to a frequent expression loss and thereby functional inactivation of the investigated genes in human breast cancer. A relevant correlation of promoter methylation with clinico-pathological parameters could be detected for ITIH5 only, which was associated with features of metastasis (positive lymph node status, higher number of positive lymph nodes and occurrence of distant metastases). In univariate survival analyses, either a methylated DKK3 or ITIH5 promoter was significantly associated with shortened recurrence-free survival. Moreover, methylation of SFRP1, SFRP5, DKK3 or ITIH5 was significantly associated with poor overall survival. More important, in multivariate survival analyses DKK3 and ITIH5 methylation emerged as strong and independent prognostic factors, able to predict early tumor recurrence and poor overall survival in human breast cancer. With regard to early tumor detection, a methylation combination of two genes (SFRP1/SFRP2) was able to detect 96% of all investigated breast carcinoma samples. Preliminary experiments aiming at detection of promoter methylation of the selected genes in patient blood serum were successfully accomplished for SFRP1, DKK3 and ITIH5. In summary, the here presented study successfully identified novel and potentially useful DNA methylation biomarkers in human breast cancer. The clinical potency of these biomarkers was characterized and the most promising candidates were suggested for further validation in independent, prospective clinical studies.","abstract_html":"Molecular biomarkers, being capable of detecting the earliest forms of a malignancy or predicting the course of a cancer disease are of constantly growing interest in modern medicine. Such biomarkers will be useful in early cancer diagnosis, as well as in the prediction of a tumor’s response to a treatment regimen, and in combination with improved prognostication may help the clinician to select the most appropriate treatment option for the benefit of a cancer patient. Since aberrant DNA methylation is essentially involved in tumorigenesis, this alteration may provide a rich source of such molecular biomarkers. In this study, six genes were analyzed for a potential utility as a DNA methylation biomarker in human breast cancer. Five of these genes (SFRP1, SFRP2, SFRP5, WIF1 und DKK3) encode secreted inhibitors of the Wnt signaling pathway, which regulates fundamental processes, such as cell proliferation and differentiation. ITIH5 encodes an extracellular matrix protein, being involved in extracellular matrix integrity and in cell adhesion. As a prerequisite, each of the selected genes contains a CpG island within the gene promoter, being a potential substrate to aberrant DNA methylation. Initially, benign and malignant breast cell lines were selected for mRNA expression analysis of the respective genes by RT-PCR/realtime PCR, and also for promoter methylation analysis by methylation-specific PCR (MSP) after genomic DNA bisulfite conversion. Malignant cell lines commonly showed increased methylation in the analyzed gene promoters in close association with downregulation of mRNA transcripts, whereas unmethylated genes exhibited abundant mRNA expression. This association was functionally confirmed, since reduced mRNA expression of each methylated gene could be significantly induced in vitro by a DNA demethylating treatment with 5-aza-2’-deoxycytidine and trichostatin A. Therefore, promoter methylation was identified as the major molecular aberration leading to a common expression loss of the analyzed genes in malignant breast cell lines. Next, promoter methylation was investigated in a large cohort of primary breast carcinomas (n=204) and corresponding normal breast tissues (n=26) by MSP. This revealed a high frequency of promoter methylation in breast carcinomas (SFRP1: 81%; SFRP2: 83%; SFRP5: 73%; WIF1: 62%; DKK3: 61%; ITIH5: 68%), whereas in normal breast tissues these genes were almost completely unmethylated. Therefore, promoter methylation of the analyzed genes represents a tumor-specific alteration in human breast cancer (P&lt;0.0001). For SFRP1, SFRP5, DKK3 and ITIH5, mRNA expression was determined in parallel to MSP for selected samples by realtime PCR. A detected frequent loss of mRNA expression (SFRP1: 87%; SFRP5: 62%; DKK3: 70%, ITIH5: 77%) was significantly associated with promoter methylation of the respective gene. Furthermore, SFRP2, DKK3 and ITIH5 expression was also quantified at the protein level by immunohistochemical staining, and revealed a frequent expression loss of SFRP2 in 75%, DKK3 in 74% and ITIH5 in 70% of the investigated breast carcinomas. Collectively, a high concordance between the frequency of promoter methylation and the frequency of expression loss could be detected for each analyzed gene. Taken together with the results from breast cell lines, promoter methylation represents the predominant molecular aberration that leads to a frequent expression loss and thereby functional inactivation of the investigated genes in human breast cancer. A relevant correlation of promoter methylation with clinico-pathological parameters could be detected for ITIH5 only, which was associated with features of metastasis (positive lymph node status, higher number of positive lymph nodes and occurrence of distant metastases). In univariate survival analyses, either a methylated DKK3 or ITIH5 promoter was significantly associated with shortened recurrence-free survival. Moreover, methylation of SFRP1, SFRP5, DKK3 or ITIH5 was significantly associated with poor overall survival. More important, in multivariate survival analyses DKK3 and ITIH5 methylation emerged as strong and independent prognostic factors, able to predict early tumor recurrence and poor overall survival in human breast cancer. With regard to early tumor detection, a methylation combination of two genes (SFRP1/SFRP2) was able to detect 96% of all investigated breast carcinoma samples. Preliminary experiments aiming at detection of promoter methylation of the selected genes in patient blood serum were successfully accomplished for SFRP1, DKK3 and ITIH5. In summary, the here presented study successfully identified novel and potentially useful DNA methylation biomarkers in human breast cancer. The clinical potency of these biomarkers was characterized and the most promising candidates were suggested for further validation in independent, prospective clinical studies.","abstract_has_math":false,"creators":["Veeck, Jürgen"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Knüchel-Clarke, Ruth"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2009,"date_issued":"2009","date_published":"2009","updated_at":"2026-07-30T19:40:25Z","subjects":["info:eu-repo/classification/ddc/610","Brustkrebs","Epigenetik","Biomarker","Methylierung","Tumorsuppressor-Gen","Prognostik","Diagnostik","Polymerase-Kettenreaktion","Real time quantitative PCR","Immuncytochemie","Kaplan-Meier-Schätzung","Blutserum","Wnt-Proteine","Proliferation","Sequenzanal","Medizin","Methylierungsspezifische PCR","MSP","Pyrosequenzierung","epigenetics","breast cancer","tumor biomarker","DNA methylation","Wnt pathway"],"languages":["ger"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113133%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113133%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113133%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/50593","outbound_label":"Repository record","outbound_source":"dc:identifier"},"source_record":{"url":"https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50593","prefix":"oai_dc"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Knüchel-Clarke, Ruth"]},{"key":"dc:creator","label":"Author","values":["Veeck, Jürgen"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2009"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-26776"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/610","Brustkrebs","Epigenetik","Biomarker","Methylierung","Tumorsuppressor-Gen","Prognostik","Diagnostik","Polymerase-Kettenreaktion","Real time quantitative PCR","Immuncytochemie","Kaplan-Meier-Schätzung","Blutserum","Wnt-Proteine","Proliferation","Sequenzanal","Medizin","Methylierungsspezifische PCR","MSP","Pyrosequenzierung","epigenetics","breast cancer","tumor biomarker","DNA methylation","Wnt pathway"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["ger"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/50593","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113133%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Molecular biomarkers, being capable of detecting the earliest forms of a malignancy or predicting the course of a cancer disease are of constantly growing interest in modern medicine. Such biomarkers will be useful in early cancer diagnosis, as well as in the prediction of a tumor’s response to a treatment regimen, and in combination with improved prognostication may help the clinician to select the most appropriate treatment option for the benefit of a cancer patient. Since aberrant DNA methylation is essentially involved in tumorigenesis, this alteration may provide a rich source of such molecular biomarkers. In this study, six genes were analyzed for a potential utility as a DNA methylation biomarker in human breast cancer. Five of these genes (SFRP1, SFRP2, SFRP5, WIF1 und DKK3) encode secreted inhibitors of the Wnt signaling pathway, which regulates fundamental processes, such as cell proliferation and differentiation. ITIH5 encodes an extracellular matrix protein, being involved in extracellular matrix integrity and in cell adhesion. As a prerequisite, each of the selected genes contains a CpG island within the gene promoter, being a potential substrate to aberrant DNA methylation. Initially, benign and malignant breast cell lines were selected for mRNA expression analysis of the respective genes by RT-PCR/realtime PCR, and also for promoter methylation analysis by methylation-specific PCR (MSP) after genomic DNA bisulfite conversion. Malignant cell lines commonly showed increased methylation in the analyzed gene promoters in close association with downregulation of mRNA transcripts, whereas unmethylated genes exhibited abundant mRNA expression. This association was functionally confirmed, since reduced mRNA expression of each methylated gene could be significantly induced in vitro by a DNA demethylating treatment with 5-aza-2’-deoxycytidine and trichostatin A. Therefore, promoter methylation was identified as the major molecular aberration leading to a common expression loss of the analyzed genes in malignant breast cell lines. Next, promoter methylation was investigated in a large cohort of primary breast carcinomas (n=204) and corresponding normal breast tissues (n=26) by MSP. This revealed a high frequency of promoter methylation in breast carcinomas (SFRP1: 81%; SFRP2: 83%; SFRP5: 73%; WIF1: 62%; DKK3: 61%; ITIH5: 68%), whereas in normal breast tissues these genes were almost completely unmethylated. Therefore, promoter methylation of the analyzed genes represents a tumor-specific alteration in human breast cancer (P<0.0001). For SFRP1, SFRP5, DKK3 and ITIH5, mRNA expression was determined in parallel to MSP for selected samples by realtime PCR. A detected frequent loss of mRNA expression (SFRP1: 87%; SFRP5: 62%; DKK3: 70%, ITIH5: 77%) was significantly associated with promoter methylation of the respective gene. Furthermore, SFRP2, DKK3 and ITIH5 expression was also quantified at the protein level by immunohistochemical staining, and revealed a frequent expression loss of SFRP2 in 75%, DKK3 in 74% and ITIH5 in 70% of the investigated breast carcinomas. Collectively, a high concordance between the frequency of promoter methylation and the frequency of expression loss could be detected for each analyzed gene. Taken together with the results from breast cell lines, promoter methylation represents the predominant molecular aberration that leads to a frequent expression loss and thereby functional inactivation of the investigated genes in human breast cancer. A relevant correlation of promoter methylation with clinico-pathological parameters could be detected for ITIH5 only, which was associated with features of metastasis (positive lymph node status, higher number of positive lymph nodes and occurrence of distant metastases). In univariate survival analyses, either a methylated DKK3 or ITIH5 promoter was significantly associated with shortened recurrence-free survival. Moreover, methylation of SFRP1, SFRP5, DKK3 or ITIH5 was significantly associated with poor overall survival. More important, in multivariate survival analyses DKK3 and ITIH5 methylation emerged as strong and independent prognostic factors, able to predict early tumor recurrence and poor overall survival in human breast cancer. With regard to early tumor detection, a methylation combination of two genes (SFRP1/SFRP2) was able to detect 96% of all investigated breast carcinoma samples. Preliminary experiments aiming at detection of promoter methylation of the selected genes in patient blood serum were successfully accomplished for SFRP1, DKK3 and ITIH5. In summary, the here presented study successfully identified novel and potentially useful DNA methylation biomarkers in human breast cancer. The clinical potency of these biomarkers was characterized and the most promising candidates were suggested for further validation in independent, prospective clinical studies."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University XI, 191 S. : Ill., graph. Darst. (2009). = Aachen, Techn. Hochsch., Diss., 2009"]},{"key":"dc:title","label":"Title","values":["Identifizierung und Charakterisierung von neuen DNA-Methylierungs-Biomarkern für das humane Mammakarzinom"]}]}],"canonical_facts":{"dc:contributor":["Knüchel-Clarke, Ruth"],"dc:coverage":["DE"],"dc:creator":["Veeck, Jürgen"],"dc:date":["2009"],"dc:description":["Molecular biomarkers, being capable of detecting the earliest forms of a malignancy or predicting the course of a cancer disease are of constantly growing interest in modern medicine. Such biomarkers will be useful in early cancer diagnosis, as well as in the prediction of a tumor’s response to a treatment regimen, and in combination with improved prognostication may help the clinician to select the most appropriate treatment option for the benefit of a cancer patient. Since aberrant DNA methylation is essentially involved in tumorigenesis, this alteration may provide a rich source of such molecular biomarkers. In this study, six genes were analyzed for a potential utility as a DNA methylation biomarker in human breast cancer. Five of these genes (SFRP1, SFRP2, SFRP5, WIF1 und DKK3) encode secreted inhibitors of the Wnt signaling pathway, which regulates fundamental processes, such as cell proliferation and differentiation. ITIH5 encodes an extracellular matrix protein, being involved in extracellular matrix integrity and in cell adhesion. As a prerequisite, each of the selected genes contains a CpG island within the gene promoter, being a potential substrate to aberrant DNA methylation. Initially, benign and malignant breast cell lines were selected for mRNA expression analysis of the respective genes by RT-PCR/realtime PCR, and also for promoter methylation analysis by methylation-specific PCR (MSP) after genomic DNA bisulfite conversion. Malignant cell lines commonly showed increased methylation in the analyzed gene promoters in close association with downregulation of mRNA transcripts, whereas unmethylated genes exhibited abundant mRNA expression. This association was functionally confirmed, since reduced mRNA expression of each methylated gene could be significantly induced in vitro by a DNA demethylating treatment with 5-aza-2’-deoxycytidine and trichostatin A. Therefore, promoter methylation was identified as the major molecular aberration leading to a common expression loss of the analyzed genes in malignant breast cell lines. Next, promoter methylation was investigated in a large cohort of primary breast carcinomas (n=204) and corresponding normal breast tissues (n=26) by MSP. This revealed a high frequency of promoter methylation in breast carcinomas (SFRP1: 81%; SFRP2: 83%; SFRP5: 73%; WIF1: 62%; DKK3: 61%; ITIH5: 68%), whereas in normal breast tissues these genes were almost completely unmethylated. Therefore, promoter methylation of the analyzed genes represents a tumor-specific alteration in human breast cancer (P<0.0001). For SFRP1, SFRP5, DKK3 and ITIH5, mRNA expression was determined in parallel to MSP for selected samples by realtime PCR. A detected frequent loss of mRNA expression (SFRP1: 87%; SFRP5: 62%; DKK3: 70%, ITIH5: 77%) was significantly associated with promoter methylation of the respective gene. Furthermore, SFRP2, DKK3 and ITIH5 expression was also quantified at the protein level by immunohistochemical staining, and revealed a frequent expression loss of SFRP2 in 75%, DKK3 in 74% and ITIH5 in 70% of the investigated breast carcinomas. Collectively, a high concordance between the frequency of promoter methylation and the frequency of expression loss could be detected for each analyzed gene. Taken together with the results from breast cell lines, promoter methylation represents the predominant molecular aberration that leads to a frequent expression loss and thereby functional inactivation of the investigated genes in human breast cancer. A relevant correlation of promoter methylation with clinico-pathological parameters could be detected for ITIH5 only, which was associated with features of metastasis (positive lymph node status, higher number of positive lymph nodes and occurrence of distant metastases). In univariate survival analyses, either a methylated DKK3 or ITIH5 promoter was significantly associated with shortened recurrence-free survival. Moreover, methylation of SFRP1, SFRP5, DKK3 or ITIH5 was significantly associated with poor overall survival. More important, in multivariate survival analyses DKK3 and ITIH5 methylation emerged as strong and independent prognostic factors, able to predict early tumor recurrence and poor overall survival in human breast cancer. With regard to early tumor detection, a methylation combination of two genes (SFRP1/SFRP2) was able to detect 96% of all investigated breast carcinoma samples. Preliminary experiments aiming at detection of promoter methylation of the selected genes in patient blood serum were successfully accomplished for SFRP1, DKK3 and ITIH5. In summary, the here presented study successfully identified novel and potentially useful DNA methylation biomarkers in human breast cancer. The clinical potency of these biomarkers was characterized and the most promising candidates were suggested for further validation in independent, prospective clinical studies."],"dc:identifier":["https://publications.rwth-aachen.de/record/50593","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113133%22"],"dc:language":["ger"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-26776"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University XI, 191 S. : Ill., graph. Darst. (2009). = Aachen, Techn. 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