Teresa Tapia

Promoter hypermethylation of BRCA1 correlates with absence of expression in hereditary breast cancer tumors.

Germline mutations in BRCA1 account for a low proportion of hereditary cases in diverse populations. Several efforts have been made to find new genes involved in the inheritance of breast cancer with no success until today. The participation of BRCA1 in the development of breast cancer has been proposed in several studies where hypermethylation of its promoter and a decrease in expression has been reported for sporadic cases and one study on familial cases. To explore the participation of BRCA1 in hereditary carcinogenesis through a different mechanism than the inheritance of germline mutations, we studied the methylation status of its promoter in breast tumors, from patients previously screened for BRCA1/BRCA2 germline mutations. We also determined the presence of the BRCA1 protein in these tumors and correlated both events with tumor grade, hormone receptors and ERBB2 presence. Promoter hypermethylation of the BRCA1 gene was detected in 51% of our biopsies, among which 67% did not express the respective protein. This result leads us to suggest that hypermethylation could be considered as an inactivating mechanism for BRCA1 expression, either as a first or second hit. Moreover, a number of biopsies with absence of expression on BRCA1 showed negative detection of estrogen and progesterone receptors, a similar phenotype to BRCA1 mutated breast tumors.

Silencing of tumor suppressor genes RASSF1A, SLIT2, and WIF1 by promoter hypermethylation in hereditary breast cancer

Promoter hypermethylation is gaining strength as one of the main mechanisms through which tumor suppressor genes are silenced during tumor progression. Three tumor suppressor genes are frequently found methylated in their promoter, in concordance with absence of expression, RASSF1A, SLIT2, and WIF1. In addition, a previous array‐CGH analysis from our group showed that these genes are found in deleted genomic regions observed in hereditary breast cancer tumors. In the present work we analyzed the methylation status of these three tumor suppressor gene promoters in 47 hereditary breast cancer tumors. Promoter methylation status analysis of hereditary breast tumors revealed high methylation frequencies for the three genes (67% RASSF1A, 80% SLIT2, and 72% WIF1). Additionally, the presence of methylated PCR products was associated with absence of protein expression for the three genes and statistically significant for RASSF1A and WIF1. Interestingly, methylation of all the three genes was found in 4 out of 6 grade I invasive ductal carcinoma tumors. Association between RASSF1A methylation and DCIS tumors was found. These results suggest that silencing of these tumor suppressor genes is an early event in hereditary breast cancer, and could be a marker for pre‐malignant phenotypes.

Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients With Gastric Cancer

Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer.

BRCA1 and BRCA2 founder mutations account for 78% of germline carriers among hereditary breast cancer families in Chile

Identifying founder mutations in BRCA1 and BRCA2 in specific populations constitute a valuable opportunity for genetic screening. Several studies from different populations have reported recurrent and/or founder mutations representing a relevant proportion of BRCA mutation carriers. In Latin America, only few founder mutations have been described. We screened 453 Chilean patients with hereditary breast cancer for mutations in BRCA1 and BRCA2. For recurrent mutations, we genotyped 11 microsatellite markers in BRCA1 and BRCA2 in order to determine a founder effect through haplotype analysis. We found a total of 25 mutations (6 novel) in 71 index patients among which, nine are present exclusively in Chilean patients. Our analysis revealed the presence of nine founder mutations, 4 in BRCA1 and 5 in BRCA2, shared by 2 to 10 unrelated families and spread in different regions of Chile. Our panel contains the highest amount of founder mutations until today and represents the highest percentage (78%) of BRCA1 and BRCA2 mutation carriers. We suggest that the dramatic reduction of Amerindian population due to smallpox and wars with Spanish conquerors, a scarce population increase during 300 years, and the geographic position of Chile constituted a favorable scenario to establish founder genetic markers in our population.Identifying founder mutations in BRCA1 and BRCA2 in specific populations constitute a valuable opportunity for genetic screening. Several studies from different populations have reported recurrent and/or founder mutations representing a relevant proportion of BRCA mutation carriers. In Latin America, only few founder mutations have been described. We screened 453 Chilean patients with hereditary breast cancer for mutations in BRCA1 and BRCA2. For recurrent mutations, we genotyped 11 microsatellite markers in BRCA1 and BRCA2 in order to determine a founder effect through haplotype analysis. We found a total of 25 mutations (6 novel) in 71 index patients among which, nine are present exclusively in Chilean patients. Our analysis revealed the presence of nine founder mutations, 4 in BRCA1 and 5 in BRCA2, shared by 2 to 10 unrelated families and spread in different regions of Chile. Our panel contains the highest amount of founder mutations until today and represents the highest percentage (78%) of BRCA1 and BRCA2 mutation carriers. We suggest that the dramatic reduction of Amerindian population due to smallpox and wars with Spanish conquerors, a scarce population increase during 300 years, and the geographic position of Chile constituted a favorable scenario to establish founder genetic markers in our population.

Different Array CGH profiles within hereditary breast cancer tumors associated to BRCA1 expression and overall survival

BackgroundArray CGH analysis of breast tumors has contributed to the identification of different genomic profiles in these tumors. Loss of DNA repair by BRCA1 functional deficiency in breast cancer has been proposed as a relevant contribution to breast cancer progression for tumors with no germline mutation. Identifying the genomic alterations taking place in BRCA1 not expressing tumors will lead us to a better understanding of the cellular functions affected in this heterogeneous disease. Moreover, specific genomic alterations may contribute to the identification of potential therapeutic targets and offer a more personalized treatment to breast cancer patients.MethodsForty seven tumors from hereditary breast cancer cases, previously analyzed for BRCA1 expression, and screened for germline BRCA1 and 2 mutations, were analyzed by Array based Comparative Genomic Hybridization (aCGH) using Agilent 4x44K arrays. Overall survival was established for tumors in different clusters using Log-rank (Mantel-Cox) Test. Gene lists obtained from aCGH analysis were analyzed for Gene Ontology enrichment using GOrilla and DAVID tools.ResultsGenomic profiling of the tumors showed specific alterations associated to BRCA1 or 2 mutation status, and BRCA1 expression in the tumors, affecting relevant cellular processes. Similar cellular functions were found affected in BRCA1 not expressing and BRCA1 or 2 mutated tumors. Hierarchical clustering classified hereditary breast tumors in four major, groups according to the type and amount of genomic alterations, showing one group with a significantly poor overall survival (p = 0.0221). Within this cluster, deletion of PLEKHO1, GDF11, DARC, DAG1 and CD63 may be associated to the worse outcome of the patients.ConclusionsThese results support the fact that BRCA1 lack of expression in tumors should be used as a marker for BRCAness and to select these patients for synthetic lethality approaches such as treatment with PARP inhibitors. In addition, the identification of specific alterations in breast tumors associated with poor survival, immune response or with a BRCAness phenotype will allow the use of a more personalized treatment in these patients.

Abstract 5077: Array CGH genomic profile of hereditary breast cancer tumors: Identification of tumor suppressor genes in deleted regions, determination of promoter hypermethylation and their protein expression in tumor biopsies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer, as other cancer types, is a genetic disease caused by sequential accumulation of mutations and genomic alterations involving tumor suppressor genes and oncogenes. In this matter, inactivation of tumor suppressor genes may occur by deletion, promoter hypermethylation or point mutations. We analyzed by array-CGH tumor DNA from 52 patients with hereditary breast cancer: 3 BRCA1, 4 BRCA2 and 45 from patients with no identified mutations (BRCAX). Our analysis revealed that BRCA1 mutated tumors have different genomic alterations compared to BRCA2 tumors. BRCAX tumors showed frequent deletions at: 1q21.3 (20%), 1p31.1 and 9q33.1 (18%); and frequent amplifications at 1q23.1 (20%), followed by 1q21.1 and several gains in chromosome 19 (18%). All genes involved in the deleted or amplified regions were compared with gene expression data in the ONCOMINE database finding correspondence among genomic alterations and gene expression. Based on these results, we selected 3 tumor suppressor genes for promoter methylation and protein expression analyses: RASSF1A, SLIT2 and WIF1. Methylation analysis through MS-PCR revealed that RASSF1A promoter was hypermethylated in 67% of hereditary tumors, and significantly associated to loss of expression (p=0.007, OR 9.6, CI 1.77-52.19). WIF1 promoter was hypermethylated in 68% of tumors with a significant association to loss of expression (p=0.042, OR 6.4, CI 1.155-35.45). Finally, SLIT2 was found as the most frequently hypermethylated promoter among tumors (80%) showing loss of expression in 90% of these tumors. Our results indicate that silencing of RASSF1A, SLIT2 and WIF1 through promoter hypermethylation may have an important role in hereditary breast tumor development. Fondecyt 1040779 y 1080595. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5077. doi:1538-7445.AM2012-5077

Abstract 2759: Possible later age of breast cancer onset for Hispanic BRCA1 carriers with the protective rs140068132-G allele

Hispanic women in the U.S. have lower incidence of breast cancer compared to Non-Hispanic Whites (NHW). A genome-wide association study (GWAS) of breast cancer in Hispanics reported a relatively strong protective variant near the ESR1 gene, only observed among women with Indigenous American ancestry (rs140068132-A/G). The study also reported lower mammographic density among women who were homozygous for the Indigenous American variant (G) and a stronger protective effect for estrogen receptor negative (ER-) breast cancer. In the present study, we assessed if this variant had an effect on age at breast cancer diagnosis among Hispanic BRCA1 carriers, who commonly present with ER- disease. We combined data from four studies of Hispanic BRCA1 mutation carriers with breast cancer: the Clinical Cancer Genetics Community Research Network (CCGCRN; N = 152), Northern California Breast Cancer Family Registry (NC-BCFR, N = 27), and two studies in Latin America, one from Colombia (N = 33) and one from Chile (N = 27). We genotyped the rs140068132 variant using a Taqman assay following the recommended protocol. We used a non-parametric Kruskal-Wallis equality-of-populations rank test to evaluate if the age of breast cancer diagnosis was associated with the rs140068132 polymorphism among BRCA1 carriers. We conducted separate analyses of Hispanic women from California and women from Colombia and Chile. For the California studies, we also had information on ovarian cancer status and were able to conduct stratified analyses. Among 239 BRCA1 carriers, we observed 201 homozygous AA, 36 heterozygous AG, and 2 missing genotypes, with an overall allele frequency of the G allele of 7.6%. We did not find a statistically significant effect of rs140068132 on age at diagnosis among Hispanic BRCA1 carriers overall. However, we found a suggestion of later age at diagnosis, with median age at diagnosis of 39.8 years (33-46 years) in AA homozygous compared to 44 years (34-50 years) in heterozygous (p value = 0.1) women from California. Among women without ovarian cancer either before or after the breast cancer diagnosis, the difference in age at breast cancer diagnosis by genotype was slightly stronger (p value 0.06). We did not observe an association among the patients from Colombia and Chile. Studies of BRCA mutation carriers are often limited by selection for breast cancer cases, thus there may be a bias against enrollment of BRCA carriers with the protective allele in the present analysis. The observed suggestion of a difference in age at diagnosis in the samples from California, similar to genome-wide identified variants that have been shown to have an effect among BRCA1 and BRCA2 carriers, warrants further investigation. Citation Format: Laura Fejerman, Jeffrey N. Weitzel, Esther M. John, Cynthia Villarreal, Gary Unzeitig, Darling Horcasitas, Charite Ricker, Adrian Daneri, Kayla Castaneda, Alexander Miron, Ana Marie Tuazon, Magdalena Echeverry, Pilar Carvallo, Carolina Alvarez, Teresa Tapia, Columbus Consortium, Luis Carvajal-Carmona, Susan Neuhausen, Elad Ziv. Possible later age of breast cancer onset for Hispanic BRCA1 carriers with the protective rs140068132-G allele. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2759. doi:10.1158/1538-7445.AM2015-2759

Abstract 2015: BRCA1 expression and genomic aberrations in triple negative breast tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Among breast cancer tumors, a group named as triple-negative breast cancer (TNBC) which do not express hormone receptors and HER2 (ER-, PR-, HER2-) do not have specific therapy. Therefore, knowing the genes involved in this type of tumor is highly relevant for treatment. Intense research in these tumors has been performed in order to identify new targets. We studied 48 TNBC tumors from Chilean patients by different approaches. In these tumors, we determined the expression, by immunohistochemistry, the different cytokeratins (CK5, CK14, and CK8/18) and EGFR. In addition, we analyzed BRCA1 expression and localization by immunohistochemistry. Using array-CGH, we characterized the genomic aberrations, in order to identify common alterations between tumors. A 62.5% (30/48) of the tumors were classified as basal-like. Expression and localization of BRCA1 analysis revealed that a 22.9% (11/48) of tumors presented normal expression of nuclear BRCA1, a 29.2% (14/48) has low or absent nuclear expression and a 47.9% (23/48) has altered BRCA1 localization, in addition to low expression in some cases. This suggests that the loss of expression of BRCA1 or its altered localization may be relevant for tumor progression. Analysis of genomic alterations by array-CGH revealed regions of gain (4p16.3-p16.2, 7p22.3-p22.2, 11p15.5, 19p13.3) and deleted regions (1q21.1, 6p11.2; 11q22.3) shared by more than 30% of these tumors. By grouping these tumors we identified three main groups, one of which correlates with the low expression / miss-localization of BRCA1. An analysis of the genes contained in these regions can determine the signaling pathways relevant to the progression of breast cancer. In this regard, by clustering, we identified a group of TNBC tumors (17/48) with amplification of AKT1, which may be relevant as a therapeutic target. FONDECYT 1080595, CONICYT 24091058. Citation Format: Teresa Tapia, Andres Aravena, Carolina Alvarez, Valeria Cornejo, Wanda Fernandez, Mauricio Camus, Alejandro Maass, Pilar Carvallo. BRCA1 expression and genomic aberrations in triple negative breast tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2015. doi:10.1158/1538-7445.AM2013-2015

Abstract 4844: Genomics aberrations and BRCA1 silencing in hereditary triple negative breast tumors in Chilean women

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Breast cancer is one of the most common cancers in women worldwide and the second cause of death by cancer among women in Chile. Either sporadic or hereditary breast cancers show a wide variety of tumor phenotypes, leading to individual clinic responses to specific treatments. Among breast cancer tumors, a group named as triple negative breast cancer (TNBC) does not express hormone receptors and HER2 (ER-, PR-, HER2-). For these tumors no specific treatment complementing chemotherapy has been developed yet. Therefore the study of the genes involved in the progression of TNBC tumor may provide a rationale for the future exploration of therapeutic strategies. TNBC is frequently observed in women carrying a germline mutation in the BRCA1 gene and in approximately 30% of non-carrier women with loss of expression of BRCA1 protein. Our aim, is to identify common genomic aberrations among TNBC tumors and to evaluate BRCA1 silencing by hypermethylation of its promoter in TNBC tumors. It has been described that about 80-90% of TNBC tumors belong to the basal-like molecular subtype, constituting a biological and clinical distinct subgroup. To determine the basal-like subtype we evaluated the expression of Cytokeratin 14 (CK14) marker in 22 hereditary TNBC. Our results revealed that a 63% (14/22) of the tumors express CK14, suggesting that these tumors belong to the basal-like subtype. Additionally, we analyzed the methylation status of the BRCA1 promoter and its correspondent protein expression in the tumors. A 77% (17/22) of the tumors had BRCA1 promoter hypermethylation. Of all tumors 32% (7/22) had absent or reduced nuclear expression of BRCA1. Interestingly, we found 54% (12/22) of the TNBC tumors with a cytoplasmic expression of BRCA1. Finally, we also analyzed through array-CGH, 19 hereditary TNBC tumors to identify common genomic aberrations between these tumors. The array-CGH analysis revealed the gain of the genomic regions 7q34, 15q26.1 and 16p13.3 and the deletion of the regions 1q21.1, 9q33.3, 18q22.1-q22.3 and 19q13.41 in more than 30% of the TNBC tumors. In conclusion, we found that 71% of tumors expressing CK14 have absent or reduced expression of the BRCA1. On the other hand, 61% of tumors with promoter hypermethylation of BRCA1 have loss expression of its protein. Interestingly, the region deleted 1q21.1 was found in 94% of the hereditary TNBC tumors. Financed by FONDECYT 1080595 and CONICYT 24091058. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4844. doi:10.1158/1538-7445.AM2011-4844