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Promoter hypermethylation of the tumor-suppressor genes ITIH5, DKK3, and RASSF1A as novel biomarkers for blood-based breast cancer screening.

Kloten V, Becker B, Winner K, Schrauder MG, Fasching PA, Anzeneder T, Veeck J, Hartmann A, Knüchel R, Dahl E - Breast Cancer Res. (2013)

Bottom Line: Based on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity.In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively.Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: For early detection of breast cancer, the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory. We investigated DNA methylation in circulating free DNA (cfDNA) from blood of breast cancer patients and matched controls to establish a biomarker panel potentially useful for early detection of breast cancer.

Methods: We examined promoter methylation of seven putative tumor-suppressor genes (SFRP1, SFRP2, SFRP5, ITIH5, WIF1, DKK3, and RASSF1A) in cfDNA extracted from serum. Clinical performance was first determined in a test set (n = 261 sera). In an independent validation set (n = 343 sera), we validated the most promising genes for further use in early breast cancer detection. Sera from 59 benign breast disease and 58 colon cancer patients were included for additional specificity testing.

Results: Based on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity. In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively. Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.

Conclusions: Tumor-specific methylation of the three-gene panel (ITIH5, DKK3, and RASSF1A) might be a valuable biomarker for the early detection of breast cancer.

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Overview of the analyzed serum samples in two independent sets and paired breast cancer tumor tissues. BBD, benign breast disease; BC, breast cancer; CC, colon cancer;N, healthy normal.
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Figure 1: Overview of the analyzed serum samples in two independent sets and paired breast cancer tumor tissues. BBD, benign breast disease; BC, breast cancer; CC, colon cancer;N, healthy normal.

Mentions: In total, 604 serum samples were assessed in this case-control study. It included samples from patients with all stages of breast cancer (n = 250 sera), cancer-free individuals (n = 237 sera), patients with benign breast disease (n = 59 sera), and patients with colon cancer (n = 58 sera). Importantly, 90% of the patients in both the test and the validation set had small tumors (pT1 or pT2). Furthermore, asymptomatic women who, based on their family history and/or mutational analysis, were suspected or proven to carry mutations in breast cancer susceptibility genes (BRCA1 and BRCA2) or had a previous breast tumor were not included in our group of healthy controls. An overview of the analyzed serum sets is summarized in Figure 1. A subset of 261 serum and 112 paired breast cancer tissue samples was used as test set, and was obtained from the tumor bank of Euregional comprehensive Cancer Center Aachen (ECCA), now part of the RWTH centralized biomaterial bank (RWTH cBMB). All patients gave informed consent for retention and analysis of their serum for research purposes (local ethical review board of the medical faculty of the RWTH Aachen, ref no. EK-206/09). The validation set consisted of 343 samples, including clinicopathologically matched samples to the test set. Samples of the validation set were obtained from the University Hospital of Erlangen and from Patients' Tumor Bank of Hope (PATH foundation, a research resource for breast cancer biosamples) [23]. Additional controls were collected from individuals with colon cancer, because colon cancer is the second common cancer type in women. In addition, various sera from benign breast disease patients, including fibroadenoma (n = 17), desmoid tumors (n = 1), benign phyllodes tumors (n = 1), mastopathy (n = 33), papilloma (n = 5), duct ectasia (n = 1), and harmatoma (n = 1) were analyzed for additional specificity testing. Additional control samples were obtained from the RWTH cBMB. An overview of the clinical characteristics of the breast cancer patients in the test and validation set is summarized in Table 1. All subjects participating were HIV, HBV, and HCV negative and had no previous history of cancer. Blood from all patients was drawn immediately or up to 2 days after diagnosis and before starting any cancer-specific treatment. All patient samples were collected between the years 2005 and 2012.


Promoter hypermethylation of the tumor-suppressor genes ITIH5, DKK3, and RASSF1A as novel biomarkers for blood-based breast cancer screening.

Kloten V, Becker B, Winner K, Schrauder MG, Fasching PA, Anzeneder T, Veeck J, Hartmann A, Knüchel R, Dahl E - Breast Cancer Res. (2013)

Overview of the analyzed serum samples in two independent sets and paired breast cancer tumor tissues. BBD, benign breast disease; BC, breast cancer; CC, colon cancer;N, healthy normal.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672828&req=5

Figure 1: Overview of the analyzed serum samples in two independent sets and paired breast cancer tumor tissues. BBD, benign breast disease; BC, breast cancer; CC, colon cancer;N, healthy normal.
Mentions: In total, 604 serum samples were assessed in this case-control study. It included samples from patients with all stages of breast cancer (n = 250 sera), cancer-free individuals (n = 237 sera), patients with benign breast disease (n = 59 sera), and patients with colon cancer (n = 58 sera). Importantly, 90% of the patients in both the test and the validation set had small tumors (pT1 or pT2). Furthermore, asymptomatic women who, based on their family history and/or mutational analysis, were suspected or proven to carry mutations in breast cancer susceptibility genes (BRCA1 and BRCA2) or had a previous breast tumor were not included in our group of healthy controls. An overview of the analyzed serum sets is summarized in Figure 1. A subset of 261 serum and 112 paired breast cancer tissue samples was used as test set, and was obtained from the tumor bank of Euregional comprehensive Cancer Center Aachen (ECCA), now part of the RWTH centralized biomaterial bank (RWTH cBMB). All patients gave informed consent for retention and analysis of their serum for research purposes (local ethical review board of the medical faculty of the RWTH Aachen, ref no. EK-206/09). The validation set consisted of 343 samples, including clinicopathologically matched samples to the test set. Samples of the validation set were obtained from the University Hospital of Erlangen and from Patients' Tumor Bank of Hope (PATH foundation, a research resource for breast cancer biosamples) [23]. Additional controls were collected from individuals with colon cancer, because colon cancer is the second common cancer type in women. In addition, various sera from benign breast disease patients, including fibroadenoma (n = 17), desmoid tumors (n = 1), benign phyllodes tumors (n = 1), mastopathy (n = 33), papilloma (n = 5), duct ectasia (n = 1), and harmatoma (n = 1) were analyzed for additional specificity testing. Additional control samples were obtained from the RWTH cBMB. An overview of the clinical characteristics of the breast cancer patients in the test and validation set is summarized in Table 1. All subjects participating were HIV, HBV, and HCV negative and had no previous history of cancer. Blood from all patients was drawn immediately or up to 2 days after diagnosis and before starting any cancer-specific treatment. All patient samples were collected between the years 2005 and 2012.

Bottom Line: Based on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity.In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively.Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: For early detection of breast cancer, the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory. We investigated DNA methylation in circulating free DNA (cfDNA) from blood of breast cancer patients and matched controls to establish a biomarker panel potentially useful for early detection of breast cancer.

Methods: We examined promoter methylation of seven putative tumor-suppressor genes (SFRP1, SFRP2, SFRP5, ITIH5, WIF1, DKK3, and RASSF1A) in cfDNA extracted from serum. Clinical performance was first determined in a test set (n = 261 sera). In an independent validation set (n = 343 sera), we validated the most promising genes for further use in early breast cancer detection. Sera from 59 benign breast disease and 58 colon cancer patients were included for additional specificity testing.

Results: Based on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity. In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively. Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.

Conclusions: Tumor-specific methylation of the three-gene panel (ITIH5, DKK3, and RASSF1A) might be a valuable biomarker for the early detection of breast cancer.

Show MeSH
Related in: MedlinePlus