Assessing Heavy Metal and PCB Exposure from Tap Water by Measuring Levels in Plasma from Sporadic Breast Cancer Patients, a Pilot Study

Breast cancer (BrCA) is the most common cancer affecting women around the world. However, it does not arise from the same causative agent among all women. Genetic markers have been associated with heritable or familial breast cancers, which may or may not be confounded by environmental factors, whereas sporadic breast cancer cases are more likely attributable to environmental exposures. Approximately 85% of women diagnosed with BrCA have no family history of the disease. Given this overwhelming bias, more plausible etiologic mechanisms should be investigated to accurately assess a woman’s risk of acquiring breast cancer. It is known that breast cancer risk is highly influenced by exogenous environmental cues altering cancer genes either by genotoxic mechanisms (DNA mutations) or otherwise. Risk assessment should comprehensively incorporate exposures to exogenous factors that are linked to a woman’s individual susceptibility. However, the exact role that some environmental agents (EA) play in tumor formation and/or cancer gene regulation is unclear. In this pilot project, we begin a multi-disciplinary approach to investigate the intersection of environmental exposures, cancer gene response, and BrCA risk. Here, we present data that show environmental exposure to heavy metals and PCBs in drinking water, heavy metal presence in plasma of nine patients with sporadic BrCA, and Toxic Release Inventory and geological data for a metal of concern, uranium, in Northeast Georgia

DARC/ACKR1 protein is differentially expressed among lymphoblasts derived from divergent ancestry groups in HAPMAP/ 1,000 (1K) Genomes populations.

<p><b>(A).</b> Representative IHC images of DARC expression in the lymphoblasts of Africans (YRI) and European Americans (CEPH/CEU). DARC is stained with Vulcan Red chromogen nuclei are stained blue with hematoxylin. <b>(B).</b> Shows a distribution analysis of IHC scores across ancestry groups and <i>Fy-</i> genotypes. The full range of scores (0–4) were observed in our cohort, with clear trends within groups. European Americans were the only ancestry group to have a 0 score, indicating no DARC expression in these cells, correlating with lower transcription of the gene. Similarly, individuals with a homozygous <i>Duffy-positive</i> (TT) genotype were the only to have a score of 0, indicating that lowest levels of DARC expression in lymphoblast are associated with the European lineage and the TT genotype, in contrast to higher DARC expression in the lymphocytes of African lineages. Accordingly, the majority of high lymphoblast IHC scores (3 or 4) were in the African lineage. All African Americans only showed moderate expression levels in lymphoblasts.</p

Statistical comparisons of relative levels of DARC isoform transcripts and ratios among ancestry and Fy- genotype groups.

<p>Statistical analyses across ancestry groups are on the left and genotype groups are on the right. ANOVA graphs indicate the individual group means (dotted blue line) of DARC isoform expression (top) and ratios (bottom) for stated categories. No statistically significant difference in individual transcript isoforms was identified across groups; however, significant differences were measured among ratios of DARC1/DARC2 isoforms. The means connection lines (solid blue and red) indicate the statistically significant comparisons. The significant p-values for pairwise T-test analyses of means is shown in the colors corresponding with each pair. The only statistically significant comparison across ancestry groups were between Amish and Yoruba groups. Across genotypes, there was a significant difference in DARC isoform ratios between TT genotype and both CT and CC genotypes. Significant differences between the isoform ratios indicate the isoforms are differentially regulated and the control of regulation is altered in isoform promoters with the C allele (Fy-).</p

The DARC gene structure and frequencies of the two major alleles; <i>Fy-</i> and <i>Fy</i>^<i>a/b</i> in HAPMAP experimental cohort compared to entire 1000 Genomes (1K) populations.

<p><b>(A).</b> Top, Model of DARC/ACKR1 gene structure indicating the two gene promoter-driven isoforms, DARC1/A and DARC2/B. Primers used for qPCR of transcript variants are indicated as double arrows. Also indicated are the <i>Duffy Null</i> allele (rs2814778) and <i>Fy</i>^<i>a/b</i> allele (rs12075). The two transcript products result in unique gene products; Duffy isoform A has 338aa and Duffy isoform B has 336aa. Top graph shows the allele frequencies of all documented polymorphisms in the DARC gene region, specifically in the ethnic cohorts of interest; African American (ASW), West African- Yoruba in Ibadan (YRI) and European American (CEPH). The overall ‘world’ variant frequencies are also indicated, summarizing all sampled populations in the 1000 Genomes. The bottom graph details the individual ancestry groups that encompass the global frequencies across the entire 1000 Genomes dataset for the entire DARC gene region. The X-axis matches the gene model with regard to relative gene structure location. The rs# IDs on the X axis of the bottom graph correspond to those on the top graph, for each SNP. The starred locus indicates a deletion polymorphism that resides in the promoter region of the DARC1/A isoform that has not previously been explicitly reported, described in the text (<i>rs17838198)</i>. This locus appears to have been under some selection influence in other populations, similar to the as the malaria selection influence on the <i>Duffy Null</i> allele in African populations, as it is the 3rd most prominent variant in the gene and prevalent in most populations. <b>(B).</b> The genotype frequencies for the <i>Fy-</i> and <i>Fy</i>^<i>a/b</i> loci in our cohort are shown in the inset panel on the left. The allele frequencies of <i>Fy-</i> and <i>Fy</i>^<i>a/b</i> for the entire corresponding ancestry 1K Genomes panels are shown in the inset panel on the right. The genotype percent distributions of the <i>Fy-</i> genotype across our West African—YRI, African American and European American subpopulations correlate with the high prevalence of the alleles in 1,000 Genome populations. Nearly half of the AA/ASW group is heterozygotes with an additional 40% being CC homozygotes, indicating more than 90% have the <i>Fy-</i> allele, as seen in the adjacent graph. The allele distribution table shows proportion values correlate with previously established numbers and validate our population.</p

Differential expression of DARC isoforms in lymphoblasts derived from 1K Genomes populations.

<p><b>(A)</b> Shows the relative expression levels of DARC 1 and DARC 2 transcripts among our HAPMAP cohort panel. The Fy- genotypes are indicated in the X axis of the top graph (A) and correlated HAPMAP cell line IDs are indicated in the X axis of the bottom graph (B). The “C” allele indicates the <i>Duffy Null</i> mutation. There is a clear trend of higher expression in the African and African Americans, with DARC 1 showing prominent expression. <b>(B)</b> Shows the relative ratios of DARC1/DARC2 isoforms in our cohort. The average ratio values for each ancestry category are shown as overlapping shaded box insets to display the values of each ancestry group. The highest ratio in the African category indicates the greatest difference between isoform expression values. C. ANOVA statistics indicate there are significant differences in DARC isoform transcripts across the cohort groups. The overall fitness across the entire group is mainly due to correlated expression of the isoforms for individuals with the C allele. There is clear trend of higher expression of the DARC 1 isoform in African and African American lineages, relative to European Americans. These data show a trend for lower expression in the European American (CEPH) categories and TT homozygotes for both DARC isoforms. These data indicate differences in isoform regulation, associated with the Fy- “<i>Duffy Null</i>” allele.</p

Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (<i>ACKR1</i>) / Duffy Antigen Receptor for Chemokines (<i>DARC</i>) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the <i>Duffy-Null</i> Allele

<div><p>The Atypical ChemoKine Receptor 1 (<i>ACKR1</i>) gene, better known as <i>Duffy</i> Antigen Receptor for Chemokines (<i>DARC</i> or <i>Duffy</i>), is responsible for the <i>Duffy</i> Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the <i>Duffy Null (Fy-)</i> allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of <i>DARC</i> which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific <i>Fy-</i> allele alters the expression pattern of <i>DARC</i> isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of <i>DARC</i> on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of <i>DARC</i> isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that <i>DARC</i> isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups.</p></div

Atypical Chemokine Receptor 1 (DARC/ACKR1) in Breast Tumors Is Associated with Survival, Circulating Chemokines, Tumor-Infiltrating Immune Cells, and African Ancestry

BACKGROUND: Tumor-specific immune response is an important aspect of disease prognosis and ultimately impacts treatment decisions for innovative immunotherapies. The atypical chemokine receptor 1 (ACKR1 or DARC) gene plays a pivotal role in immune regulation and harbors several single-nucleotide variants (SNV) that are specific to sub-Saharan African ancestry. METHODS: Using computational The Cancer Genome Atlas (TCGA) analysis, case-control clinical cohort Luminex assays, and CIBERSORT deconvolution, we identified distinct immune cell profile-associated DARC/ACKR1 tumor expression and race with increased macrophage subtypes and regulatory T cells in DARC/ACKR1-high tumors. RESULTS: In this study, we report the clinical relevance of DARC/ACKR1 tumor expression in breast cancer, in the context of a tumor immune response that may be associated with sub-Saharan African ancestry. Briefly, we found that for infiltrating carcinomas, African Americans have a higher proportion of DARC/ACKR1-negative tumors compared with white Americans, and DARC/ACKR1 tumor expression is correlated with proinflammatory chemokines, CCL2/MCP-1 (P \u3c0.0001) and anticorrelated with CXCL8/IL8 (P \u3c0.0001). Sub-Saharan African-specific DARC/ACKR1 alleles likely drive these correlations. Relapse-free survival (RFS) and overall survival (OS) were significantly longer in individuals with DARC/ACKR1-high tumors (P \u3c1.0 x 10(-16) and P \u3c2.2 x 10(-6), respectively) across all molecular tumor subtypes. CONCLUSIONS: DARC/AKCR1 regulates immune responses in tumors, and its expression is associated with sub-Saharan African-specific alleles. DARC/ACKR1-positive tumors will have a distinct immune response compared with DARC/AKCR1-negative tumors. IMPACT: This study has high relevance in cancer management, as we introduce a functional regulator of inflammatory chemokines that can determine an infiltrating tumor immune cell landscape that is distinct among patients of African ancestry

Atypical Chemokine Receptor 1 (DARC/ACKR1) in Breast Tumors Is Associated with Survival, Circulating Chemokines, Tumor-Infiltrating Immune Cells, and African Ancestry

BACKGROUND: Tumor-specific immune response is an important aspect of disease prognosis and ultimately impacts treatment decisions for innovative immunotherapies. The atypical chemokine receptor 1 (ACKR1 or DARC) gene plays a pivotal role in immune regulation and harbors several single-nucleotide variants (SNV) that are specific to sub-Saharan African ancestry. METHODS: Using computational The Cancer Genome Atlas (TCGA) analysis, case-control clinical cohort Luminex assays, and CIBERSORT deconvolution, we identified distinct immune cell profile-associated DARC/ACKR1 tumor expression and race with increased macrophage subtypes and regulatory T cells in DARC/ACKR1-high tumors. RESULTS: In this study, we report the clinical relevance of DARC/ACKR1 tumor expression in breast cancer, in the context of a tumor immune response that may be associated with sub-Saharan African ancestry. Briefly, we found that for infiltrating carcinomas, African Americans have a higher proportion of DARC/ACKR1-negative tumors compared with white Americans, and DARC/ACKR1 tumor expression is correlated with proinflammatory chemokines, CCL2/MCP-1 (P \u3c0.0001) and anticorrelated with CXCL8/IL8 (P \u3c0.0001). Sub-Saharan African-specific DARC/ACKR1 alleles likely drive these correlations. Relapse-free survival (RFS) and overall survival (OS) were significantly longer in individuals with DARC/ACKR1-high tumors (P \u3c1.0 x 10(-16) and P \u3c2.2 x 10(-6), respectively) across all molecular tumor subtypes. CONCLUSIONS: DARC/AKCR1 regulates immune responses in tumors, and its expression is associated with sub-Saharan African-specific alleles. DARC/ACKR1-positive tumors will have a distinct immune response compared with DARC/AKCR1-negative tumors. IMPACT: This study has high relevance in cancer management, as we introduce a functional regulator of inflammatory chemokines that can determine an infiltrating tumor immune cell landscape that is distinct among patients of African ancestry

Atypical Chemokine Receptor 1 (DARC/ACKR1) in Breast Tumors Is Associated with Survival, Circulating Chemokines, Tumor-Infiltrating Immune Cells, and African Ancestry

BACKGROUND: Tumor-specific immune response is an important aspect of disease prognosis and ultimately impacts treatment decisions for innovative immunotherapies. The atypical chemokine receptor 1 (ACKR1 or DARC) gene plays a pivotal role in immune regulation and harbors several single-nucleotide variants (SNV) that are specific to sub-Saharan African ancestry. METHODS: Using computational The Cancer Genome Atlas (TCGA) analysis, case-control clinical cohort Luminex assays, and CIBERSORT deconvolution, we identified distinct immune cell profile-associated DARC/ACKR1 tumor expression and race with increased macrophage subtypes and regulatory T cells in DARC/ACKR1-high tumors. RESULTS: In this study, we report the clinical relevance of DARC/ACKR1 tumor expression in breast cancer, in the context of a tumor immune response that may be associated with sub-Saharan African ancestry. Briefly, we found that for infiltrating carcinomas, African Americans have a higher proportion of DARC/ACKR1-negative tumors compared with white Americans, and DARC/ACKR1 tumor expression is correlated with proinflammatory chemokines, CCL2/MCP-1 (P \u3c0.0001) and anticorrelated with CXCL8/IL8 (P \u3c0.0001). Sub-Saharan African-specific DARC/ACKR1 alleles likely drive these correlations. Relapse-free survival (RFS) and overall survival (OS) were significantly longer in individuals with DARC/ACKR1-high tumors (P \u3c1.0 x 10(-16) and P \u3c2.2 x 10(-6), respectively) across all molecular tumor subtypes. CONCLUSIONS: DARC/AKCR1 regulates immune responses in tumors, and its expression is associated with sub-Saharan African-specific alleles. DARC/ACKR1-positive tumors will have a distinct immune response compared with DARC/AKCR1-negative tumors. IMPACT: This study has high relevance in cancer management, as we introduce a functional regulator of inflammatory chemokines that can determine an infiltrating tumor immune cell landscape that is distinct among patients of African ancestry