Ana Estrada

Genome-wide association study of breast cancer in Latinas identifies novel protective variants on 6q25

The genetic contributions to breast cancer development among Latinas are not well understood. Here we carry out a genome-wide association study of breast cancer in Latinas and identify a genome-wide significant risk variant, located 5′ of the Estrogen Receptor 1 gene (ESR1; 6q25 region). The minor allele for this variant is strongly protective (rs140068132: odds ratio (OR) 0.60, 95% confidence interval (CI) 0.53–0.67, P=9 × 10−18), originates from Indigenous Americans and is uncorrelated with previously reported risk variants at 6q25. The association is stronger for oestrogen receptor-negative disease (OR 0.34, 95% CI 0.21–0.54) than oestrogen receptor-positive disease (OR 0.63, 95% CI 0.49–0.80; P heterogeneity=0.01) and is also associated with mammographic breast density, a strong risk factor for breast cancer (P=0.001). rs140068132 is located within several transcription factor-binding sites and electrophoretic mobility shift assays with MCF-7 nuclear protein demonstrate differential binding of the G/A alleles at this locus. These results highlight the importance of conducting research in diverse populations.

The HABP2 G534E Variant Is an Unlikely Cause of Familial Nonmedullary Thyroid Cancer

CONTEXT A recent study reported the non-synonymous G534E (rs7080536, allele A) variant in the HABP2 gene as causal in familial non-medullary thyroid cancer (NMTC). OBJECTIVE The objective of this study was to evaluate the causality of HABP2 G534E in the TCUKIN study, a multi-center population based study of NMTC cases from the British Isles. DESIGN AND SETTING A case-control analysis of rs7080536 genotypes was performed using 2,105 TCUKIN cases and 5,172 UK controls. PARTICIPANTS Cases comprised 2,105 NMTC cases. Patients sub-groups with papillary (N=1,056), follicular (N=691) and Hurthle cell (N=86) TC cases were studied separately. Controls comprised 5,172 individuals from the 1958 Birth Cohort (58C) and the National Blood Donor Service (NBS) study. The controls had previously been genotyped using genome-wide SNP arrays by the Wellcome Trust Case Control Consortium study. OUTCOME Measures: Association between HABP2 G534E (rs7080536A) and NMTC risk was evaluated using logistic regression. RESULTS The frequency of HABP2 G534E was 4.2% in cases and 4.6% in controls. We did not detect an association between this variant and NMTC risk (OR=0.896, 95% CI: 0.746-1.071, P=0.233). We also failed to detect an association between HABP2 G534E and cases with papillary (1056 cases, G534E frequency= 3.5%, OR=0.74, P=0.017), follicular (691 cases, G534E frequency= 4.7%, OR=1.00, P=1.000) or Hurthle cell (86 cases, G534E frequency= 6.3%, OR=1.40, P=0.279) histology. CONCLUSIONS We found that HABP2 G534E is a low-to-moderate frequency variant in the British Isles and failed to detect an association with NMTC risk, independent of histological type. Hence, our study does not implicate HABP2 G534E or a correlated polymorphism in familial NMTC and additional data are required before using this variant in NMTC risk assessment.

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.

The HABP2 G534E polymorphism does not increase nonmedullary thyroid cancer risk in Hispanics

Familial nonmedullary thyroid cancer (NMTC) has not been clearly linked to causal germline variants, despite the large role that genetic factors play in risk. Recently, HABP2 G534E (rs7080536A) has been implicated as a causal variant in NMTC. We have previously shown that the HABP2 G534E variant is not associated with TC risk in patients from the British Isles. Hispanics are the largest and the youngest minority in the United States and NMTC is now the second most common malignancy in women from this population. In order to determine if the HABP2 G534E variant played a role in NMTC risk among Hispanic populations, we analyzed 281 cases and 1105 population-matched controls from a multicenter study in Colombia, evaluating the association through logistic regression. We found that the HABP2 G534E variant was not significantly associated with NMTC risk (P=0.843) in this Hispanic group. We also stratified available clinical data by multiple available clinicopathological variables and further analyzed the effect of HABP2 on NMTC presentation. However, we failed to detect associations between HABP2 G534E and NMTC risk, regardless of disease presentation (P≥0.273 for all cases). Therefore, without any significant associations between the HABP2 G534E variant and NMTC risk, we conclude that the variant is not causal of NMTC in this Hispanic population.

The 8q24 rs6983267G variant is associated with increased thyroid cancer risk.

The G allele of the rs6983267 single-nucleotide polymorphism, located on chromosome 8q24, has been associated with increased risk of several cancer types. The association between rs6983267G and thyroid cancer (TC) has been tested in different populations, mostly of European ancestry, and has led to inconclusive results. While significant associations have been reported in the British and Polish populations, no association has been detected in populations from Spain, Italy and the USA. To further investigate the role of rs6983267G in TC susceptibility, we evaluated rs6983267 genotypes in three populations of different continental ancestry (British Isles, Colombia and Japan), providing a total of 3067 cases and 8575 controls. We detected significant associations between rs6983267G and TC in the British Isles (odds ratio (OR)=1.19, 95% CI: 1.11-1.27, P=4.03×10(-7)), Japan (OR=1.20, 95% CI: 1.03-1.41, P=0.022) and a borderline significant association of similar effect direction and size in Colombia (OR=1.19, 95% CI: 0.99-1.44, P=0.069). A meta-analysis of our multi-ethnic study and previously published non-overlapping datasets, which included a total of 5484 cases and 12 594 controls, confirmed the association between rs6983267G and TC (P=1.23×10(-7), OR=1.13, 95% CI: 1.08-1.18). Our results therefore support the notion that rs6983267G is a bona fide TC risk variant that increases the risk of disease by ∼13%.

Abstract 42: Development of low-cost high-throughput screening methods for detecting germline mutations in multiple cancer genes

Cancer is a leading cause of non-communicable morbidity and mortality worldwide. While many genes that predispose individuals for different cancers have been discovered, the population prevalence of mutations in these genes remains largely undetermined in many populations. Developing customized screening panels and screening for novel variants for a large population samples can be expensive and time consuming. We have developed a low-cost high-throughput pipeline and method to screen 480 customizable amplicons (∼20 genes, ∼144Kbp) for up to 384 samples per run. By combining a bioinformatics pipeline for design and analysis with Fluidigm microfluidics PCR and Illumina MiSeq, we can quickly screen the full coding sequence of multiple cancer panels at a high depth of coverage at a low cost per sample (∼

Abstract 1786: One in four Hispanic women with early onset breast cancer carry BRCA1, BRCA2, or PALB2 mutations: Results from a population-based study in South America

20-40/sample). The amplicon design pipeline provides ability to develop amplicon primer sets and pooling strategies. Libraries of 384 barcoded samples are run in a single MiSeq lane. Sequencing data is analyzed by an automated pipeline, which aligns using BWA-MEM, calls variants using VarScan 2, and annotates variants using multiple datasets using Annovar. We have applied these pipelines and methods to identify mutation in known cancer genes, such as APC, MSH2, MLH1, BRCA1, and BRCA2, in several hundred Hispanic individuals with familial and early-onset colon and breast cancer. While many of these mutations have been previously reported, we have identified several novel pathogenic changes that appear to have Amerindian origin. Of which, a few are shared among many individuals in isolated geographic locations, suggesting founder effects are common in some of these populations. In conclusion, we have developed a low-cost high-throughput method for screening customizable panels for known and novel mutations. These panels typically consist of 20 cancer genes and our group has already developed panels that are specific for breast cancer, colon cancer and thyroid cancer. Our study is an initial step to assess prevalence of known cancer causing genes and identify novel genes/mutations that contribute to different cancers in the Hispanic community. These discoveries provide a foundation for early detection, prevention, and treatment of familial cancers. Citation Format: Ruta Sahasrabudhe,, Paul Lott, Anna Marie Tuazon, John Williamson, Natalia Belter, Ana Estrada, Mabel Bohorquez, Rodrigo Prieto, Angel Criollo, Alejandro Velez, Jorge Castro, Gilbert Mateus, Magdalena Echhevery, Luis G. Carvajal-Carmona. Development of low-cost high-throughput screening methods for detecting germline mutations in multiple cancer genes. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 42. doi:10.1158/1538-7445.CANSUSC14-42

Abstract 2784: Genetic analysis of breast cancer in admixed populations of central Colombia

Breast cancer is the leading cause of cancer incidence and mortality among Hispanic women, who are often diagnosed with late stage tumors and are more likely to die after diagnosis when compared to non-Hispanic whites. While strides have been made in understanding Hispanic breast cancer genetics, most studies have been limited to investigating cases from high-risk clinics. In this study, we aimed to assess the prevalence of pathogenic mutations in three most important breast cancer genes (BRCA1, BRCA2 and PALB2) in an unselected Hispanic breast cancer cohort. Initially, six hundred and forty-six unselected Colombian breast cancer cases were screened for four known Hispanic BRCA1/2 mutations by genotyping. Subsequently, cases that remained mutation negative, as well as 186 cancer-free controls, were screened for BRCA1, BRCA2 and PALB2 mutations using targeted next generation sequencing. We identified 67 cases with pathogenic mutations, one case with a likely-pathogenic variant, and 16 carriers of variants of unknown significance. Among the pathogenic mutation carriers, 88.1% harbored a founder mutation (n = 59), which includes the known BRCA1 3450del4 mutation, strikingly found in 32 unrelated cases. Remarkably, we found that 1 in 4 of the cases diagnosed with breast cancer by age 40 years, regardless of family history of cancer, carried a pathogenic mutation. The high frequency of pathogenic mutations in this unselected cohort (10.4%), in particular those with an early age of onset (25.3%), suggests that population-based genetic testing among Hispanic communities can identify most carriers who would otherwise ineligible for testing. Identifying mutation carriers of these genes has implications in clinical management and surveillance for Hispanic women, a population that is vulnerable to breast cancer disparities in the U.S. and Latin America. Citation Format: Anna Marie Tuazon, Mabel Bohorquez, Carolina Ramirez, Paul Lott, Ana Estrada, Angel Criollo, Cathy Wang, Magdalena Echeverry, John Suarez, COLUMBUS Consortium, LaFamilia Consortium, Luis G. Carvajal Carmona. One in four Hispanic women with early onset breast cancer carry BRCA1, BRCA2, or PALB2 mutations: Results from a population-based study in South America. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1786.

Abstract 2739: Transcontinental characterization of the Hispanic BRCA1 3450del4 breast cancer founder mutation

Breast cancer (BC) is the most frequently diagnosed cancer among women with an estimated 1,676,633 new cases resulting in ∼521,817 deaths each year globally. Thus, it is no surprise BC research has become one of the hottest cancer research areas in the past three decades. Past studies examining and identifying BC risk loci have focused on three major populations (European, African, and Asian). Past research has led to greater understanding of the etiology (genetic and environmental) and treatment of BC; resulting in a ∼30% decrease in mortality rate in wealthy countries. However, when looking at BC incidence and mortality in low to middle income nations similar gains are not seen; in fact, in these countries mortality has increased in the past 20 years. This trend holds true for the Hispanic community. While Hispanic women have a ∼27% lower rate of BC than in non-Hispanic white women, Hispanic women are more likely to be diagnosed with larger tumors and later stage cancer, leading to ∼20% increase in mortality for Hispanic women compared to their non-Hispanic white counterpart with similar diagnosis stage and age. Differences in incidence and mortality have been attributed to environmental and genetic factors. This case-control association study aims to address a research disparity by examining the role of ethnicity and genetics on development of BC risk in central Colombian Hispanic populations. It provides one of the first assessments of known BC risk factors in the South American Hispanic community. The study was conducted in central Colombia using ∼600 breast cancer cases from COLUMBUS initiative & ∼700 matched cancer-free controls from the CHIBCHA consortium. Cases were genotyped using Affymetrix Axiom UK Biobank arrays and controls were genotyped using both Affymetrix Axiom LAT and Axiom Custom arrays followed by imputation using 1000 Genomes data. We analyzed previously discovered BC loci and under independent analysis found 6 significant loci: rs11780156 (P = 0.005, OR: 1.34), rs2981582 (P = 0.007, OR: 1.25), rs4784227 (P = 0.016, OR: 1.22), rs2981579 (P = 0.017, OR 1.21), rs1011970 (P = 0.0274, OR 1.20), rs1219648 (P = 0.042, OR 1.18) and several loci showing borderline significant associations involving the same risk allele. Interestingly, we identified additional novel BC risk loci that were genome-wide significant and are undergoing replication in populations from Central Mexico. In summary, we performed an assessment of BC risk loci in central Colombian Hispanic population and found significant overlap with BC risk loci previously identified in European and East Asian populations as well as potentially novel loci that are unique to Hispanic populations. These results are integral to the establishment of personalized medicine programs and will form the basis for future BC prevention and treatment program in Latin American and US Hispanics. Citation Format: Paul C. Lott, Angel Criollo, Anna Marie Tuazon, Carolina Ramirez Alfonso, Ruta Sahasrabudhe, Ana Estrada, Elisha Garcia, Rodrigo Prieto, John Williamson, Javier Torres, Mabel Bohorquez, Maria Magdalena Echeverry de Polanco, CHIBCHA Consortium, COLUBMUS Consortium, Luis G. Carvajal-Carmona. Genetic analysis of breast cancer in admixed populations of central Colombia. [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 2784. doi:10.1158/1538-7445.AM2015-2784

Abstract 1520: Development of low-cost high throughput screening pipeline for detecting germline cancer causing mutations in Hispanic populations

Breast cancer (BC) is the most commonly diagnosed cancer among women, with 1 in every 7 women in the U.S. developing BC in their lifetime. While BC may not be the leading cause of cancer incidence in many minority populations, such as Hispanics, it is the leading cause of cancer death, highlighting a cancer disparity. Moreover, the contribution of mutations in BC susceptibility genes remains poorly investigated in Hispanic populations. To begin to address these voids, we screened Colombian BC patients for common BRCA1 and BRCA2 mutations using the KASP genotyping system. Strikingly, 27 of 722 unselected cases (4%) harbored the same BRCA1 3450del4 mutation. Clinical data and family history indicate that these individuals originate from a distinct geographic region in Colombia, Neiva. Significantly, 10% of the unselected BC cases in Neiva and the surrounding region harbor the 3450del4 mutation, potentially representing one of the most profound founder effects reported in human populations. Interesting, this BRCA1 mutation has also been reported in other countries, including Portugal, Spain, and Chile. To determine whether this mutation stems from a single origin, mutation carriers from each of these countries (n = 26), along with mutation carriers from Colombia (n = 27), were subjected to the Affymetrix Axiom Human UK Biobank Array (>800 thousand markers). Haplotypes in which the BRCA1 mutation resides were estimated by SHAPEIT, and pairwise segmental sharing was determined using GERMLINE. Our data suggest that the mutation carriers, although from different countries and continents, share a core haplotype, likely to have originated from Spain. Additionally, the mutation seems to be fairly new to Colombia, with individuals sharing up to a 40Mb haplotype comprising the mutation. We are currently estimating the age of the mutation using the bioinformatic software DMLE+, which exploits the rate of linkage disequilibrium decay due to recombination. By understanding the prevalence of mutations in known breast cancer risk genes in minority populations, cancer disparities can be better addressed and breast cancer screening can be improved. Furthermore, these findings have direct implications in robust and cost-effective targeted screening in regions where there is a high prevalence of the BRCA1 3450del4 mutation. Citation Format: Anna Marie De Asis Tuazon, Carolina Ramirez, Mabel Bohorquez, Rodrigo Prieto, Paul Lott, Angel Criollo, Ana Estrada, Gillbert Mateus, Alejandro Velez, Justo Ramirez, COLUMBUS Consortium, Manuel Teixeira, Ana Vega, Conxi Lazaro, Eva Tornero, Cristina Martinez, Mar Infante, Miguel De La Hoya, Orland Diez, Pilar Carvallo, Magdalena Echeverry, Luis Carvajal-Carmona. Transcontinental characterization of the Hispanic BRCA1 3450del4 breast cancer founder mutation. [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 2739. doi:10.1158/1538-7445.AM2015-2739