Blanca Torres-Torres

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

BackgroundTumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients.MethodsPatients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique).ResultsOur results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively.ConclusionSilencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.

P1-05-06: Hypermethilation 14-3-3- Sigma Promoter as a Biomarker to Screening for Metastasis and Potencial Prognostic Factor in Breast Cancer Patients.

Background: 14-3-3sigma is an epithelial marker whose expression is induced by DNA damage through a p53-dependent pathway. 14-3-3sigma functions sequesters cyclin B1-CDC2 complexes outside the nucleus and thereby contributes to a G2 arrest. Epigenetic silencing by CpG methylation, p53 inactivation, and proteasome-dependent proteolysis leads to loss of 14-3-3sigma and is often observed in precancerous lesions and likely to be causally linked to the onset of cancer Objetive: To correlation methylation levels of promoter 14-3-3 sigma with association prognostic factors in breast cancer and their correlation with phenotype luminal. Material and Methods: This is a prospective study we quantified methylation levels of promoter 14-3-3 sigma gene in 107 women with breast cancer and 108 control subjects by Real Time QMS-PCR SYBR green (methylation-specific PCR) and analyzed association with prognostics factor in breast cancer. Results: Median age was 58 years (32-88); 69% were postmenopausal women. Nodal involvement N0; 63%,N1;30%,N2;7%), tumor size (T1;58%,T2;35%,T3;4%,T4;4%) and grade G1; 20%,G2;37%,G3;30%). Significant differences between breast cancer patients (pts) and healthy controls in relative serum levels of methylated gene promoters 14-3-3s (p=0.0047) were detected. Presence of methylated 14-3-3-s in serum of breast cancer patients was associated with T3-4 stage (OMS) (p 0.05). Conclusions: Hypermethylation of the 14-3-3 sigma promoter is an early and frequent event in breast neoplastic transformation, leading to the suggestion that silencing of 14-3-3 sigma may be an important event in tumor progression and particularly in breast carcinogenesis. This study identifies the presence of variations in global levels of methylation promoters genes in patients breast cancer with different phenotype classes and shows that these differences have clinical significance. Perhaps in the detection of CpG methylation of 14-3-3sigma may be used for diagnostic and prognostic purposes. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-05-06.

Abstract A47: Study of promoter methylation pattern of 14-3-3 sigma gene in serum of patients with breast cancer: A potential biomarker for diagnostic anf prognostic factor.

Background: Expression of 14-3-3 σ is a tumor suppressor gene induced in response to DNA damage, and has been implicated in G2/M cell cycle arrest by p53. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism for gene inactivation. Objective: To correlation methylation levels of promoter 14-3-3σ with association prognostic factors in breast cancer. Material and Methods: This is a prospective study we quantified methylation levels of promoter 14-3-3σ gene in 107 women with breast cancer and 108 control subjects by real time QMS-PCR SYBR green and analyzed association with prognostics factor in breast cancer. Results: Median age was 58 years (32-88); 69% were postmenopausal women.Nodal involvement N0;63%,N1;30%,N2;7%), tumor size (T1;58%,T2;35%,T3;4%,T4;4%) and grade G1; 20%, G2; 37%, G3; 30%). The methylation of 14-3-3σ were 60% of sporadic breast cancer patients and were 34% of normal breast (p=0.0047).The methylation of 14-3-3σ gene in serum was markedly related with T3-4 stage (p 0.05). Conclusions: Hypermethylation of the 14-3-3σ a promoter is an early and frequent event in breast neoplastic transformation, leading to the suggestion that silencing of 14-3-3σ may be an important event in tumor progression and particularly in breast carcinogenesis.Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Perhaps in the detection of CpG methylation of 14-3-3σ may be used for diagnostic and prognostic purposes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A47. Citation Format: Martinez-Galan Joaquina, Juan Ramon Delgado, Blanca Torres-Torres, Rosario Del Moral, Sandra Rios, M. Isabel Nunez. Study of promoter methylation pattern of 14-3-3 sigma gene in serum of patients with breast cancer: A potential biomarker for diagnostic anf prognostic factor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A47.

Abstract A126: Change in status promoter of 5 panel gene during progression of atypical hyperplasia to breast cancer.

Background: DNA methylation is the main epigenetic modification in cancer. The pathological features of breast cancer follow a sequential progression from transition of a normal cell to benign proliferative hyperplasia, hyperplasia with atypia, in situ carcinoma and, eventually, to invasive and metastatic disease. Purpose: This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions to invasive ductal carcinoma and different methylation patron with benign breast disease. Material and Methods: We performed analysis for the methylation status of 5 promoter CpG island (ESR1, E-Cad, APC, RARB, and 14-3-3-; promoters) in a group with women with normal breast tissue n=74; other group with epithelial atypia/ atypical ductal hyperplasia (n=20), ductal carcinoma in situ (DCIS, n=14) and invasive ductal carcinoma (IDC, n=107). Results: Relative serum levels of methylated gene promoters ESR1 and 14-3-3-; showed significant differences between IDC y normal breast tissue (were significantly higher in IDC than normal breast tissue) and between normal breast tissue and epithelial atypia/ atypical ductal hyperplasia/ ductal carcinoma in situ. There are no differences between IDC and epithelial atypia/ atypical ductal hyperplasia/ ductal carcinoma in situ when 14-3-3-; and ESR1 mean values are compared. In fact, the present findings showed a frequent presence of methylated 14-3-3-; in sera from women with breast benign disease, which could be considered the result of a proliferative alteration of epithelial and stromal components of the breast. Which may indicate presence of occult benign breast disease, although other possible sources of this DNA include normal tissues, which show higher methylation values with increasing age; which might undergo lysis during tumor growth; or premalignant lesion during tumor growth; or premalignant lesion. Conclusions: These findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and breast cancer, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A126. Citation Format: Martinez-Galan Joaquina, Juan Ramon Delgado, Blanca Torres-Torres, Rosario Del Moral, Sandra Rios, M. Isabel Nunez. Change in status promoter of 5 panel gene during progression of atypical hyperplasia to breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A126.

Abstract A67: Methylation of ESR1 and 14-3-3 sigma promoter in serum as possible biomarkers for screening and diagnosis for breast cancer

Background: To investigate the association between gene hypermethylation and the major clinico-pathological features of breast cancer, including diagnosis and treatment response. Material and Methods: 107 women with breast cancer and 108 control subjects were recruited. Real Time QMS-PCR SYBR green (methylation-specific PCR) was used to analyze the utility of circulating DNA with CpG island hypermethylation of APC, RAR-β, E-Cadherin, ESR1 and 14-3-3 sigma; gene promoter regions as breast cancer biomarkers. Sera were collected in 107 operable breast cancer patients (pts) previously surgery and in 60 of those pts after treatment. Respect controls, 34 had benign breast disease and 74 with no evidence of breast disease. Results: We found significant differences between breast cancer patients and healthy controls in relative serum levels of methylated gene promoters ESR1 (p = 0.0112) and 14-3-3sigma (p=0.0047). We observed that hypermethylation of these combined two genes differentiated between breast cancer patients and healthy controls (p <0.0001) with a sensitivity of 81% (95% CI: 72–88%) and specificity of 88% (95% CI: 78–94 %). Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p = 0.0179); and presence of methylated 14-3-3-sigma was associated with T3-4 stage (OMS) (p< 0.05) and nodal positive status (p< 0.05). We observed lower methylated ERS1 or 14-3-3-sigma values after surgery, respect pretreatment levels, but without an overall statistically significant difference. With a median follow up of 6 years, we found that patients with a significant decrease of sera methylated levels of both genes after surgery had better time to progression an overall survival respect patients without this observation. Conclusions: this panel of genes detected in ductal lavage and blood specimens could be useful as markers for early detection of breast carcinoma. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA is a promising tool for cancer risk assessment. Note: This abstract was not presented at the conference. Citation Format: Joaquina Martinez-Galan, Blanca Torres-Torres, Juan Ramon Delgado. Methylation of ESR1 and 14-3-3 sigma promoter in serum as possible biomarkers for screening and diagnosis for breast cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A67.

The prognostic value of detection methylation level panel of gene in DNA circulating after and before treatment in patients with breast cancer.

67 Background: Objective of this study was to determine the concordance of promoter methylation of 14-3-3σ and ESR1 in circulating DNA of breast cancer patients with response and their association with clinicopathological parameters and disease prognosis. METHODS Plasmawas sampled prospectively from 110 patients diagnosed of breast cancer. A PCR quantitative technique was used to analyze the utility of circulating DNA with CpG island hypermethylation of ESR1, 14-3-3σ, Rar-B and APC genes promoter regions as breast cancer biomarkers. RESULTS The cutoff points for the genes methylated promoters were established from the ROC curves, selecting values that gave the maximal likelihood ratio. Presence of methylated ESR1 in serum of breast cancer patients was associated with ER-negative phenotype (p=0.0179); and presence of methylated 14-3-3σ was associated with T3-4 stage (p<0.05) and nodal positive status (p<0.05). Mean serum values of methylated genes before treatment was for ESR1:0.009µg/ml, 14-3-3σ:0.047µg/ml, Rar B:0.0001µg/ml and APC:0.012µg/ml. Mean serum values of methylated genes after treatment was for ESR1:0.003 µg/ml, 14-3-3σ:0.038µg/ml, Rar-B:0 µg/ml and APC:0.001µg/ml. In the light of the discriminatory power of ESR1 and 14-3-3σ and the finding that serum levels of methylated gene promoters changed after breast cancer treatment, post-treatment modifications in these two promoters were analyzed. We observed lower methylated ERS1,14-3-3-σ and APC values after surgery, respect pretreatment levels, but without an overall statistically significant difference.With a median follow-up of 8 years, we found that patients with a significant decrease of sera methylated levels of these genes after surgery had better time to progression an overall survival respect patients without this observation. CONCLUSIONS These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA is a promising tool for cancer prognostic assessment.

Implications prognostics of methylation 14-3-3 sigma promoter in peripheral blood cell DNA with nodal involvement status and tumor size for breast cancer patients.

33 Background: Expression of 14-3-3 σ is a tumor suppressor gene induced in response to DNA damage, and has been implicated in G2/M cell cycle arrest by p53. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism for gene inactivation. To correlation methylation levels of promoter 14-3-3σ with association prognostic factors in breast cancer. METHODS This is a prospective study we quantified methylation levels of promoter 14-3-3σ gene in 107 women with breast cancer and 108 control subjects by Real Time QMS-PCR SYBR green and analyzed association with prognostics factor in breast cancer. RESULTS Median age was 58 years (32-88); 69% were postmenopausal women. Nodal involvement N0; 63%,N1;30%,N2;7%), tumor size (T1;58%,T2;35%,T3;4%,T4;4%) and grade G1; 20%,G2;37%,G3;30%). The methylation of 14-3-3σ were 60% of sporadic breast cancer patients and were 34% of normal breast (p=0.0047). The methylation of 14-3-3σ gene in serum was markedly related with T3-4 stage (p<0.05),nodal positive status (p<0.05) and poor outcome. With a median follow up 6 years we saw more probability of developing distance metastasis in patients with methylation 14-3-3σ (p>0.05). CONCLUSIONS Hypermethylation of the 14-3-3σ a promoter is an early and frequent event in breast neoplastic transformation, leading to the suggestion that silencing of 14-3-3σ may be an important event in tumor progression and particularly in breast carcinogenesis.Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Perhaps in the detection of CpG methylation of 14-3-3σ may be used for diagnostic and prognostic purposes.

Abstract B09: PROMOTER CPG ISLAND METHYLATION DURING PROGRESSION OF ATYPICAL HYPERPLASIA TO BREAST CANCER

DNA methylation is the main epigenetic modification in cancer. The pathological features of breast cancer follow a sequential progression from transition of a normal cell to benign proliferative hyperplasia, hyperplasia with atypia, in situ carcinoma and, eventually, to invasive and metastatic disease. Purpose: This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions to invasive ductal carcinoma and different methylation patron with benign breast disease. Material and Methods: We performed analysis for the methylation status of 5 promoter CpG island (ESR1, E-Cad, APC, RARB and 14-3-3-σ promoters) in a group with women with normal breast tissue n=74; other group with epithelial atypia/ atypical ductal hyperplasia (n=20), ductal carcinoma in situ (DCIS, n=14) and invasive ductal carcinoma (IDC, n=107). Results:Relative serum levels of methylated gene promoters ESR1 and 14-3-3-σ showed significant differences between IDC y normal breast tissue (were significantly higher in IDC than normal breast tissue) and between normal breast tissue and epithelial atypia/ atypical ductal hyperplasia/ ductal carcinoma in situ. There are no differences between IDC and epithelial atypia/ atypical ductal hyperplasia/ ductal carcinoma in situ when 14-3-3-σ and ESR1 mean values are compared. In fact, the present findings showed a frequent presence of methylated 14-3-3-σ in sera from women with breast benign disease, which could be considered the result of a proliferative alteration of epithelial and stromal components of the breast. Which may indicate presence of occult benign breast disease, although other possible sources of this DNA include normal tissues, which show higher methylation values with increasing age; which might undergo lysis during tumour growth; or premalignant lesion during tumour growth; or premalignant lesion. Conclusions: These findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and breast cancer, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression. Citation Format: Joaquina Martinez-Galan, Juan Ramon Delgado, Rosario Del Moral, Blanca Torres-Torres. Promoter CpG island methylation during progression of atypical hyperplasia to breast cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B09.

Methylation RE in serum of patients with breast cancer and relation with molecular phenotype and worse prognostic factors.

224 Background: To determine whether Estrogen Receptor (ESR1) (+) and ESR1(-) status relates to epigenetic changes in breast cancer-related genes and to correlate with molecular breast cancer subtypes. METHODS Since January/02 to June/05, we quantified methylation levels ERS1 gene in serum of 92 pts breast cancer. A PCR quantitative technique was used to analyze levels of methylation gene. We also examined and correlationed the expression of ESR1 in tumors by immunohistochemistry with molecular phenotype. RESULTS Median age was 58 years (32-88); 69% were postmenopausal women. Nodal involvement (N0; 63%, N1; 30%, N2; 7%), tumor size (T1; 58%, T2; 35%, T3; 4%, T4; 4%) and grade (G1; 20%, G2; 37%, G3; 30%). Of the cases, 37 pts (40%) were Luminal A (LA), 32 pts (33%) Luminal B (LB), 14 pts (15%) Triple-negative (TN) and 9pts (10%) HER2+. The methylated ESR1 in serum was significantly associated with ESR1(-) in breast tumors >80% (p=0.0179). Methylation ESR1 was preferably associated with TN (80%) and HER2+ (60%) subtype. Nevertheless unmethylation ESR1 was found more frequently in LA (71%) and LB (59%) phenotype. With a median follow up of 5 years, we found worse overall survival (OS) with more frequent ESR1 methylation gene (p>0.05), Luminal A; ESR1 Methylation OS at 5 years 81% vs 93% when was ESR1 Unmethylation. Luminal B; ESR1 Methylation 86% SG at 5 years vs 92% in Unmethylation ESR1. Triple negative; ESR1 Methylation SG at 5 years 75% vs 80% in unmethylation ESR1. HER2; ESR1 Methylation SG at 5 years was 66.7% vs 75% unmethylation ESR1. CONCLUSIONS Gene promoter region hypermethylation is a significant event in primary breast cancer. However, its impact on tumor progression and potential predictive implications remain relatively unknown. Our study identifies the presence of variations in global levels of methylation promoters ESR1 genes in breast cancer with different phenotype classes and shows that these differences have clinical significance. Although numerous issues remain to be resolved, quantitative measurement of circulating methylated DNA may be of significance in the assessment and search of targeted therapy resistance related to ESR1 and HER2 status by epigenetic or transcriptional cancer therapy.

Correlation of serum ESR1 methylation levels with RE status in tumor and molecular subtypes of breast cancer.

e13648 Background: To determine whether estrogen receptor (ER) (+) and ER (-) status relates to epigenetic changes in breast cancer-related genes and to correlate with molecular breast cancer subtypes. Methods: From January 2002 to June 2005, we quantified methylation levels ERS1 gene in serum of 92 pts breast cancer. A PCR quantitative technique was used to analyze levels of methylation gene. We also examined and correlationed the expression of ESR1 AND HER2+ in tumors by immunohistochemistry with molecular phenotype. Results: Median age was 58 years (32-88); 69% were postmenopausal women. Nodal involvement (N0;63%,N1;30%,N2;7%), tumor size (T1;58%, T2;35%, T3;4%, T4;4%) and grade (G1;20%, G2;37%, G3;30%). Of the cases, 37pts (40%) were Luminal A (LA), 32pts (33%) Luminal B (LB), 14pts (15%) triple-negative (TN), and 9pts (10%) HER2+. The methylated ESR1 in serum was significantly associated with ER(-) in breast tumors >80% (p=0.0179). Methylation ESR1 was preferably associated with TN (80%) and HER2+ (6...