Mauricio Camus

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.

Correlation between Ki67 and Histological Grade in Breast Cancer Patients Treated with Preoperative Chemotherapy

BACKGROUND AND AIM Breast cancer (BC) is a heterogeneous disease and cell proliferation markers may help to identify subtypes of clinical interest. We here analyzed the correlation between cell proliferation determined by Ki67 and HG in BC patients undergoing preoperative chemotherapy (PCT). MATERIALS AND METHODS We obtained clinical/pathological data from patients with invasive BC treated at our institution from 1999 until 2012. Expression of estrogen receptor (ER), progesterone receptor (PR), epidermal growth factor receptor type 2 (HER2) and Ki67 were determined by immuno-histochemistry (IHC). Clinicopathological subtypes were defined as: Luminal A, ER and/or PR positive, HER2 negative, HG 1 or 2; Luminal B, ER and/or PR positive, HER2 negative or positive and/or HG 3; triple negative (TN), ER, PR and HER2 negative independent of HG; HER2 positive, ER, PR negative and HER2 positive, independent of HG. By using Ki67, a value of 14% separated Luminal A and B tumors, independently of the histological grade. We analyzed correlations between Ki67 and HG, to define BC subtypes and their predictive value for response to PCT. RESULTS 1,560 BC patients were treated in the period, 147 receiving PCT (9.5%). Some 57 had sufficient clinicopathological information to be included in the study. Median age was 52 years (26-72), with 87.7% invasive ductal carcinomas (n=50). We performed IHC for Ki67 in 40 core biopsies and 50 surgical biopsies, 37 paired samples with Ki67 before and after chemotherapy being available. There was no significant correlation between Ki67 and HG (p=0.237), both categorizing patients into different subtypes. In most cases Ki67 decreased after PCT (65.8%). Only 3 patients had pathologic complete response (cPR). CONCLUSIONS In our experience we did not find associations between Ki67 and HG. Determination of clinicopathological luminal subtypes differs by using Ki67 or HG.

Breast cancer and pregnancy: a comparative analysis of a Chilean cohort

Introduction Recent reports show that pregnancy-associated breast cancer (PABC) survival is similar to that of non-pregnant young patients. We evaluate the characteristics and prognosis of PABC patients treated in our cancer centre. Patients and methods We identified patients with invasive PABC who were treated between 1999 and May 2013 and compared their characteristics with a no PABC cohort of similar age. Results The prevalence of PABC was 1% (n = 17). The median age was 35 years (range: 29– 42 years). The initial tumour was suspected clinically in 93% of the cases. Total mastectomy rates were higher in women with PABC (78.6% versus 40.5%, p = 0.02), and more tumours in the PABC group were triple negative, epidermal growth factor type 2 (HER2)–positive, and at advanced stages; however, these differences were not statistically significant. While estimated overall survival at ten years was higher in the non-PABC group (75.5% versus 80.5%, p = 0.043), disease-specific survival (DSS) rate at ten years was not statistically different between groups (83.9% for PABC and 75.5% for unrelated pregnancy BC, p = 0.37). Conclusions PABC is a rare event. In our cohort, it tended to be more aggressive. Compared with a similar age cohort, the DSS was not worse.

How much survival benefit is necessary for breast cancer patients to opt for adjuvant chemotherapy? Results from a Chilean survey

Background: Breast cancer (BC) is the leading cause of cancer death in Chilean women. Adjuvant chemotherapy decreases recurrence and death from BC. The recommendation to indicate chemotherapy is complex. Adjuvant! Online is a valuable computational tool to predict survival benefit obtained with adjuvant systemic therapy. Previous studies in Caucasian patients with BC showed that they are willing to receive chemotherapy for a small benefit. No studies, to our knowledge, have been done in the Hispanic or Latino populations. Methods: We interviewed females with BC who had previously received adjuvant chemotherapy. Age, stage at presentation, time since last chemotherapy, type of chemotherapy, marital status, number of children, and level of education were recorded. We used the graphic representation from Adjuvant! Online to question each patient on how much survival benefit she required to accept chemotherapy. Results: There were 101 women surveyed. The average age was 55.9 (±10.2), 54.5% had involved lymph nodes, 59.4% were married, and 15.8% did not have parity; 62.3% of females accepted chemotherapy for an absolute survival benefit of 1% or less. In a multivariate analysis, younger (p = 0.02) and less-educated patients (p = 0.018) were associated with lower survival benefit required to opt for chemotherapy. Conclusion: In our study, the acceptance of chemotherapy by the Hispanic population requires minimal survival benefit and is in agreement with the Caucasian population reported elsewhere. To our knowledge, our report is the first study that evaluates the perception of Latino patients regarding the benefit of chemotherapy in early BC.

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.

Evaluación del valor pronóstico de la relación neutrófilos/linfocitos en cáncer de mama de subtipos agresivos

Background: The white blood cell count is one of the most sensitive markers associated with inflammation. The neutrophil/lymphocyte count ratio may be an independent factor for breast cancer mortality. Aim: To assess the predictive value of the neutrophil/lymphocyte ratio for mortality in breast cancer. Material and methods: Review of the database of a cancer center of a University hospital. Patients with infiltrating breast cancer treated between 1997 and 2012 were selected. The pathology type and lymph node involvement were obtained from the pathology report. The expression of estrogen, progesterone and Human Epidermal Growth Factor Receptor 2 (HER2) was determined by immunohistochemistry or in situ fluorescent hybridization (FISH). The absolute peripheral neutrophil and lymphocyte counts were obtained from a complete blood count obtained at least three months before treatment. Patients were followed for a median of 61 months (range 1-171). Results: From 323 eligible patients, after excluding those in stage IV and those without an available complete blood count, 131 patients were analyzed (81 with negative receptors and 117 HER2 enriched). The neutrophil/lymphocyte ratio was similar in both type of tumors (2.1 and 1.91 respectively). Twenty two patients died during follow up. Surviving patients with HER2 enriched tumors had a lower neutrophil/lymphocyte ratio than those who died (1.79 and 3.21 respectively, p < 0.01). In a multivariate analysis, including age, tumor stage and lymph node involvement as confounding factors, the neutrophil/lymphocyte ratio was still significantly associated with a risk of death with a hazard ratio of 2.56. Conclusions: A high neutrophil/lymphocyte ratio in the complete blood count can be a predictor of death in breast cancer.

Breast Cancer at Extreme Ages - a Comparative Analysis in Chile

BACKGROUND Young onset breast cancer (BC) has a worse outcome as compared to in the elderly. However, some studies have shown that BC in the elderly, despite indolent features, does also cause increase in mortality. In an attempt to compare clinic-pathological characteristics, BC subtypes and survival in patients with BC presenting at extremes of age, we performed a retrospective study. MATERIALS AND METHODS Patients were either ≤40 or ≥70 years old. Subtypes were defined using immunohistochemistry and histological grade. Chi-Square test was used for evaluation of categorical variables, and Kaplan-meier and log-rank for disease-specific survival (DSS) and disease free survival (DFS) . RESULTS We analyzed 256 patients ≤40 and 366 patients ≥70. Younger patients presented with more aggressive disease, with less luminal A but more luminal B and triple negative (TN) subtype. With a median follow-up of 57.5 months, DFS at 5 years in younger patients was 72.3% vs 84.6% in the elderly (p=0.007). Luminal A and B disease presented with worse DFS in younger patients. The opposite was seen in the TN subgroup. Although we found no significant differences in DSS, older patients with TN tumors died of BC more frequently. This group also received less chemotherapy. CONCLUSIONS Young patients present with more aggressive disease, this translating into worse DFS. However, elderly patients with TN disease represent a particular subpopulation with worse DFS and DSS, suggesting that chemotherapy should not be withheld only because of age.

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