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Women of sub-Saharan African descent have disproportionately higher incidence of triple-negative breast cancer (TNBC) and TNBC-specific mortality across all populations. Population studies show racial differences in TNBC biology, including higher prevalence of basal-like and quadruple-negative subtypes in African Americans (AA). However, previous investigations relied on self-reported race (SRR) of primarily U.S. populations. Due to heterogeneous genetic admixture and biological consequences of social determinants, the true association of African ancestry with TNBC biology is unclear. To address this, we conducted RNA sequencing on an international cohort of AAs, as well as West and East Africans with TNBC. Using comprehensive genetic ancestry estimation in this African-enriched cohort, we found expression of 613 genes associated with African ancestry and 2,000+ associated with regional African ancestry. A subset of African-associated genes also showed differences in normal breast tissue. Pathway enrichment and deconvolution of tumor cellular composition revealed that tumor-associated immunologic profiles are distinct in patients of African descent.Significance:Our comprehensive ancestry quantification process revealed that ancestry-associated gene expression profiles in TNBC include population-level distinctions in immunologic landscapes. These differences may explain some differences in race–group clinical outcomes. This study shows the first definitive link between African ancestry and the TNBC immunologic landscape, from an African-enriched international multiethnic cohort.See related commentary by Hamilton et al., p. 2496.This article is highlighted in the In This Issue feature, p. 2483

Supplementary Table from African Ancestry–Associated Gene Expression Profiles in Triple-Negative Breast Cancer Underlie Altered Tumor Biology and Clinical Outcome in Women of African Descent

Supplementary Data from African Ancestry–Associated Gene Expression Profiles in Triple-Negative Breast Cancer Underlie Altered Tumor Biology and Clinical Outcome in Women of African Descent

Women of sub-Saharan African descent have disproportionately higher incidence of triple-negative breast cancer (TNBC) and TNBC-specific mortality across all populations. Population studies show racial differences in TNBC biology, including higher prevalence of basal-like and quadruple-negative subtypes in African Americans (AA). However, previous investigations relied on self-reported race (SRR) of primarily U.S. populations. Due to heterogeneous genetic admixture and biological consequences of social determinants, the true association of African ancestry with TNBC biology is unclear. To address this, we conducted RNA sequencing on an international cohort of AAs, as well as West and East Africans with TNBC. Using comprehensive genetic ancestry estimation in this African-enriched cohort, we found expression of 613 genes associated with African ancestry and 2,000+ associated with regional African ancestry. A subset of African-associated genes also showed differences in normal breast tissue. Pathway enrichment and deconvolution of tumor cellular composition revealed that tumor-associated immunologic profiles are distinct in patients of African descent. Significance: Our comprehensive ancestry quantification process revealed that ancestry-associated gene expression profiles in TNBC include population-level distinctions in immunologic landscapes. These differences may explain some differences in race–group clinical outcomes. This study shows the first definitive link between African ancestry and the TNBC immunologic landscape, from an African-enriched international multiethnic cohort. See related commentary by Hamilton et al., p. 2496. This article is highlighted in the In This Issue feature, p. 2483

Women of sub-Saharan African ancestry have disproportionately higher incidence of aggressive, early-onset Triple Negative Breast Cancer (TNBC), and TNBC mortality across all race groups. Population-based comparative studies show racial differences in TNBC tumor biology, with higher prevalence of basal-like and Quadruple-Negative subtypes in African Americans (AA). However, most investigations relied on self-reported race (SRR) of primarily United States (US) populations. However, given that genetic admixture in AAs is extremely heterogenous, and race-correlated social determinants can translate into biological differences, the true association of African ancestry with TNBC biology and gene expression is currently unclear. To address this, we conducted RNAseq on an international cohort of AAs, west and east Africans with TNBC. Using genetic ancestry estimation in this African-enriched cohort, we identified 613 genes associated with African ancestry and more than 2200 genes associated with regional-level African ancestry. Functional enrichment and deconvolution revealed tumor-associated immune cell infiltration and activity.STATEMENT OF SIGNIFICANCEUsing a rigorous ancestry quantification process, we show that TNBC has ancestry-associated gene expression profiles, linked to immunological landscapes, which may contribute to racial differences in clinical outcomes. This is the first study to show the definitive link to tumor immunological landscape, associated with African ancestry, using a multiethnic African-enriched cohort.

DARC/ACKR1 erythrocyte expression, also known as the “Duffy blood group,” is understood to sequester pro-inflammatory chemokines and thereby regulate circulating gradients that direct immune cell infiltration. We hypothesize that this function also determines immune cell landscapes in the tumor microenvironment. Due to evolutionary selection pressures of malaria, individuals with sub-Saharan African ancestry typically carry the Duffy-null allele (rs4849887) and this lack of DARC/ACKR1 expression gives immunity to malaria while also allowing chronically high inflammation levels. Over 68% of African Americans (AA) have been found to have the Duffy-null genotype, compared to a rare 1-3% in European American individuals and we have shown it increases predisposition for Triple-Negative Breast Cancer (TNBC). In addition, we are currently studying if Duffy-null may contribute to higher breast cancer mortality that disproportionately affects AA women. This may be in part due to the role that low-DARC/ACKR1 expression plays in chronic inflammation, altering levels of several chemokines that modulate the migration and differentiation of specific immune cells. This role will impact tumor immune cell infiltration as well as the immune cell population composition overall, depending upon levels of DARC/ACKR1. Using RNA sequencing, our initial results indicated that for breast cancer tumors with high DARC/ACKR1 expression there was a higher estimated presence of CD8+ T cells, CD4+ T cells, regulatory T cells, follicular helper T cells, and memory B cells. Whereas with low DARC/ACKR1 expression, there was markedly less expression of resting dendritic cells and memory B cells. Therefore, in order to ascertain the influence DARC status has on spatial deposition and functional status of immune cell landscapes across the tumor microenvironment, we performed imaging mass cytometry on primary TNBC tumors. The panel contained tumor, structural, and immune markers, and was used to characterize the spatial differences between samples that had been verified to be DARC-high or DARC-low through immunohistochemistry. Our imaging analyses indicated that high DARC/ACKR1 expression correlates with infiltration of monocytes, macrophages, and cytotoxic T cells into the solid tumor microenvironment. Conversely, tumors with low DARC/ACKR1 expression showed monocytes and cytotoxic T cells contained in the tumor stroma. Using single-cell phenotyping, we were also able to identify distinct cell populations between DARC-high and -low. The tSNE analysis and heatmaps performed using Histology Topography Cytometry Analysis Toolbox (histoCAT), allowed us to visualize the spatial distribution of these cell populations, indicating an immune-suppressive tumor microenvironment in DARC-low tumors. These differences may be implicated in the causality of tumor progression as well as how to approach treatment given the cell heterogeneity of TNBC. This work provides greater context on the role that Duffy-null plays in chronic inflammation on the tumor microenvironment. Citation Format: Yanira Guerra, Rachel Martini, Jessica Bensenhaver, Yalei Chen, Joseph K. Oppong, Ishmael Kyei, Frances S. Aitpillah, Michael O. Adinku, Joseph K. Oppong, Ernest K. Adjei, Aisha Jibril, Baffour Awuah, Mahteme Bekele, Engida Abebe, Kwasi Ankomah, Ernest B. Osei-Bonsu, Kofi K. Gyan, Clayton Yates, Kim Blenman, Olivier Elemento, Lisa Newman, Melissa B. Davis. The role of African Duffy-null allele related inflammation on the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-141.