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Research Article

Atypical Chemokine Receptor 1 (DARC/ACKR1) inBreast Tumors Is Associated with Survival,Circulating Chemokines, Tumor-InfiltratingImmune Cells, and African AncestryBrittany D. Jenkins1, Rachel N. Martini1, Rupali Hire1, Andrea Brown1, Briana Bennett1,I'nasia Brown1, Elizabeth W. Howerth2, Mary Egan3, Jamie Hodgson3, Clayton Yates4,Rick Kittles5, Dhananjay Chitale6, Haythem Ali7, David Nathanson8, Petros Nikolinakos3,Lisa Newman8, Michele Monteil9, and Melissa B. Davis1,9,10

Abstract

Background:Tumor-specific immune response is an impor-tant aspect of disease prognosis and ultimately impacts treat-ment decisions for innovative immunotherapies. The atypicalchemokine receptor 1 (ACKR1 or DARC) gene plays a pivotalrole in immune regulation and harbors several single-nucleotide variants (SNV) that are specific to sub-SaharanAfrican ancestry.

Methods: Using computational The Cancer Genome Atlas(TCGA) analysis, case–control clinical cohort Luminex assays,and CIBERSORT deconvolution, we identified distinctimmune cell profile–associated DARC/ACKR1 tumor expres-sion and race with increased macrophage subtypes and regu-latory T cells in DARC/ACKR1-high tumors.

Results: In this study, we report the clinical relevance ofDARC/ACKR1 tumor expression in breast cancer, in thecontext of a tumor immune response that may be associatedwith sub-Saharan African ancestry. Briefly, we found that forinfiltrating carcinomas, African Americans have a higherproportion of DARC/ACKR1-negative tumors compared with

white Americans, and DARC/ACKR1 tumor expression iscorrelated with proinflammatory chemokines, CCL2/MCP-1(P <0.0001) and anticorrelated with CXCL8/IL8 (P <0.0001).Sub-Saharan African-specific DARC/ACKR1 alleles likely drivethese correlations. Relapse-free survival (RFS) and overallsurvival (OS) were significantly longer in individualswith DARC/ACKR1-high tumors (P <1.0 � 10�16 andP <2.2 � 10�6, respectively) across all molecular tumorsubtypes.

Conclusions:DARC/AKCR1 regulates immune responses intumors, and its expression is associated with sub-SaharanAfrican-specific alleles. DARC/ACKR1-positive tumors willhave a distinct immune response compared with DARC/AKCR1-negative tumors.

Impact: This study has high relevance in cancer manage-ment, as we introduce a functional regulator of inflammatorychemokines that can determine an infiltrating tumor immunecell landscape that is distinct among patients of Africanancestry.

IntroductionBreast cancerdisparitieshavebeenwell-documented forover50

years, indicating significant differences in rates of incidence andmortality among women of varying self-reported race and ethnic-ity (1, 2). While the focus of disparities research has traditionallybeen studied through a lens of health care and/or socioeconomicbarriers (3), we and others have recently identified additionalbiological factors that are associated with disparate clinical out-comes, including disproportionate burdens of aggressive tumorsubtypes (refs. 4–8; i.e., triple-negative basal-like) in women ofwest African descent (9–12). Overall, women of African descentare more likely to be diagnosed with the worst types of breastcancer, including triple-negative breast cancer (TNBC), at earlierageswith thepoorestprognosis indications (8,10,13–15), despitehaving access to proper screening and standard treatments (16).There is a clear need to better understand how the moleculardynamics of tumor progression may differ among women ofAfrican descent, compared with women of European descent.

1Department of Genetics, Franklin College of Arts and Sciences, University ofGeorgia, Athens, Georgia. 2Department of Pathology, College of VeterinaryMedicine, University of Georgia, Athens, Georgia. 3University Cancer and BloodCenter, Athens, Georgia. 4Department of Biology and Center for CancerResearch, Tuskegee University, Tuskegee, Alabama. 5Department of PopulationSciences, City of Hope Comprehensive Cancer Center, Duarte, California.6Department of Pathology, Henry Ford Health System, Detroit, Michigan.7Department of Hematology and Oncology, Henry Ford Health System, Detroit,Michigan. 8Department of Surgery, Henry Ford Health System, Detroit, Michigan.9Department of Molecular Biology and Biochemistry, Augusta University/Uni-versity of Georgia Medical Partnership, Athens, Georgia. 10Department of PublicHealth Sciences, Henry Ford Health System, Detroit, Michigan.

Note: Supplementary data for this article are available at Cancer Epidemiology,Biomarkers & Prevention Online (http://cebp.aacrjournals.org/).

Corresponding Author: Melissa B. Davis, Weill Cornell Medicine, 1300 YorkAvenue, New York, NY 10065. Phone: 646-962-4273; Fax: 646-962-0023;E-mail: [email protected]

doi: 10.1158/1055-9965.EPI-18-0955

�2019 American Association for Cancer Research.

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Some evidence of these differences in pathologic/histologictumor variables indicate that breast cancer in women of Africanancestry has a distinct genetic signature (4, 17) and distinctassociations with immune responses (18–20) even within theTNBC subtype. These differences are the likely causes forincreased prevalence of refractory tumors in certain patientgroups. With the advent of genomic technologies and increasedgenetic diversity in multi-ethnic studies, differences in thesetumor characteristics can now be investigated to fully compre-hend dynamics of ancestry-related immunologic interactionsand how immunotherapy may or may not work for certaingroups (21). Recent studies have begun to directly address thisquestion from the perspective of genetic risk (22); however, wesought to identify key biological pathways that are systemicallyaltered in West African populations, which may drive immu-nologic differences in tumor biology in these patients. In termsof genetic risk, the typical focus of genetic background studies,these genetic drivers might not alter tumor etiology, per se, butrather tumor phenotypes. We utilized data from The CancerGenome Atlas (TCGA) to uncover expression pattern differ-ences in immunologic pathway genes that were associated withself-identified race and identified an enrichment in chemokinesignaling. The study presented here describes our in-depthinvestigation of a key regulator of these chemokine signalingpathways, atypical chemokine receptor 1 (ACKR1, aka DARC),in a breast cancer case–control cohort.

The DARC/ACKR1 is a seven-transmembrane G-protein–coupled receptor that is found on the surface of red bloodcells (RBC)/erythrocytes, endothelial cells lining postcapillaryvenules, and most recently has been shown to be expressed onlymphoblast cells (7). DARC/ACKR1 functions as a decoyreceptor for a variety of CXC and CC chemokines, includingthose with promalignant and proinflammatory effects, such asCCL2 and CXCL8 (IL8; ref. 23). It is considered a decoy receptorbecause it binds and internalizes these chemokines, and targetsthem for lysosomal degradation, efficiently sequestering theirsignaling capabilities (24, 25). In addition, DARC/ACKR1receptor has also been shown to regulate transcytosis of thebound ligands in endothelial cells (26), influencing leukocytetrafficking and the ability of the receptor to maintain homeo-static chemokine levels (27). Erythrocyte-specific DARC/ACKR1 expression plays a key role in inflammation by seques-tering chemokines from circulation to recover homeostaticlevels during inflammatory responses (26). A population-private DARC/ACKR1 gene mutation, defined as DARC/ACKR1es (erythrocyte silent), removes expression of DARC/ACKR1,specifically on erythrocytes, and is also known as the exten-sively characterized "Duffy-null blood group" (28, 29). Thismutation is mostly restricted to populations of West Africandescent and remains fixed (100% allele frequency) in present-day populations within west and central sub-Saharan Africa.

Our study defines a set of significant clinical associations ofDARC/ACKR1 among patient demographics, population-private alleles, and tumor phenotype characteristics that pro-vide greater insight to ancestry-specific differences in tumorbiology, particularly immunologic responses that are associat-ed with clinical outcomes and that may ultimately inform thedecisions for oncologic use of immunotherapy treatments (19,20, 30). We anticipate that this research will aid in decreasingthe tumor-subtype disparities in incidence and mortalityamong race groups.

Materials and MethodsEthics statement, study design, biospecimens, and cohortsummary

This study is a human subjects study and all biospecimens usedin this study were obtained after approval from one of twoinstitutional review board (IRB)-approved protocols from eitherthe University of Georgia (Athens, GA; IRB ID: MOD00003730)or Henry Ford Health System (Detroit, MI; IRB ID: 4825). Allresearch was performed in accordance with the IRB guidelines, inaccordance with a filed assurance of the IRB by the U.S. Depart-ment of Health and Human Services. All participants wereinformed of the study purpose and procedures and have signedan informed consent prior to participation and donation ofblood, saliva, and/or tissue for this study.

Gene expression and DARC/ACKR1 subtype analysesGene expression levels were obtained from RNA sequencing

(RNAseq) data accessed through the web-portal of The CancerGenome Atlas Breast Cancer cohort [n ¼ 838, 167 African Amer-icans (AA), 671 White Americans (WA); https://cancergenome.nih.gov]. After filtering samples for tissue status (removing sam-ples that were normal tissue and metastatic tumors) and histo-logic findings (removing samples that were annotated as "other,"metaplastic, or noninfiltrating), we conducted linear regressionanalyses for gene correlations, stratified as indicated for specificcontexts of interactions (i.e., molecular subtypes/phenotypes)and nominal logistical regressions across derived tumor status.For DARC/ACKR1 subtype analyses, DARC/ACKR1 expression(Supplementary Fig. S1A) was stratified on the basis of quartileranking and shown as high (upper 30th quartile), medium(intermediate quartile), and low (lower 30th) categories (Sup-plementary Fig. S1B). DARC/ACKR1 subtypes were then mea-sured for associations with specific demographic or clinical vari-ables by stratifying the population according to these variables(e.g., race and molecular breast cancer subtype; SupplementaryFig. S1C and S1D) and comparing distributions ofDARC/ACKR1subtypes within or among each category. We conducted multi-variate modeling to assess effect estimates and adjust for demo-graphic variables (i.e., race and age).

Cytokine analysis with DARC/ACKR1 subtypesThe UCSC Xena Browser (http://xenabrowser.net, accessed

April 2018) was used to generate a heatmap of TCGA breast-invasive carcinoma RNAseq gene expression data (IlluminaHi-Seq, n¼ 399) compared with a user-generated geneset of relevantcytokine genes (n¼ 67, Supplementary Table S1). DichotomizedDARC/ACKR1-positive (red) and -negative (blue) subgroupsweredetermined according to the values in Supplementary Table S2.P values for select cytokines were determined using Welch t test.

Blood and serum specimensFollowing informed consent, and at time of enrollment or time

of surgery, approximately 4 mL of blood was collected in EDTA-treated vacutainer tubes (BD Biosciences) from each newly diag-nosedpatient pretreatment atUniversityCancer andBloodCenter(Athens, GA; n ¼ 41) and Henry Ford Health System (HFHS,Detroit, MI; n ¼ 225). Blood samples from noncancer controls(n ¼ 67) and breast cancer survivors (n ¼ 17) were collected in asimilar manner at the time of enrollment at the University ofGeorgia (UGA) Clinical and Translational Research Unit (CTRU,

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Athens, GA; n¼ 84). All blood samples were processed within 24hours of collection. Undiluted plasma was collected throughcentrifugation, and the remaining sample was separated via aFicoll–Paque gradient (GE Healthcare) according to the manu-facturer's instructions. Serum control samples from premeno-pausal (n¼ 31) and postmenopausal women (n¼ 30) and breastcancer survivors (n ¼ 11) were purchased from the Susan G.Komen Tissue Bank (n ¼ 72). A flowchart detailing patientnumbers and study exclusions can be found in SupplementaryFig. S2.

Luminex human chemokine multiplex assayThe multiplex assay was completed using patient undiluted

plasma and serum. Plasma chemokine levels were quantifiedusing the Bio-Plex Pro Human Chemokine Assay Kit (Bio-Rad)that was custom designed to measure the following chemokinesand cytokines: CCL2/MCP-1, CXCL8/IL-8, CXCL9/MIG, CXCL1/Gro-a, and IL10. The assay was carried out following manufac-turer's instructions, and the results were analyzed using the Bio-Plex Manager Software version 6.1.1. Statistical multivariate pair-wise correlation analyses with these analytes can be found inSupplementary Table S3.

Red blood cell phenotyping by immunofluorescenceRBCs isolated from Ficoll–Paque blood separation techniques

arefixed in4%paraformaldehyde and stainedwithDARC/ACKR1goat anti-human (Novus Biologicals) primary antibody andAlexaFluor 488 chicken anti-goat (Invitrogen) fluorescent secondaryantibody using standard immunofluorescence techniques. Posi-tive plasma membrane control stains were done using CellMaskPlasma Membrane Stain (Life Technologies) according to themanufacturer's instructions. RBCs were imaged using a KeyenceBZ fluorescent microscope at 40� magnification.

Duffy-null genotypingGenotyping for the Duffy-Null SNV, rs2814778, was per-

formed using the TaqMan GTXpress Master Mix and variant-specific probes (Applied Biosystems) according to the manufac-turer's instructions. The assay was executed using an AppliedBiosystems 7500 Fast Real-Time PCR System. Cohort allelic andgenotypic frequencies compared with global populations from1000 Genomes data via ENSEMBL (www.ensembl.org) areprovided in Supplementary Table S4.

Tumor-associated leukocytes' gene profilingThe CIBERSORT online platform (31) was used to estimate the

absolute fractions of 22 leukocyte populations in TCGA samplesdenoted as breast primary tumor samples. The analysis was runwith 500 permutations, and quantile normalization disabled (asrecommended by the tool for RNAseq data). Only those sampleswith a maximum significance value of P < 0.05 were included inthe final analysis (n ¼ 472).

Survival analysesPatient survival associations with gene expression data were

calculated from 3,951 patients with breast cancer. The data (plat-forms: Affymetrix Microarrays: HG-U133A, HG-U133 Plus 2.0,and HG-U133A 2.0) were downloaded from Gene ExpressionOmnibus (GEO) and Array Express (AE) databases (ref. 32;Supplementary Table S5). Recurrence-free survival (RFS) andoverall survival (OS)was assessed across all breast cancer subtypes

and treatment types with dichotomized grouping for expressionofDARC/ACKR1 (probe ID: 208335_s_at), CCL2 (216598_s_at),and CXCL8 (211506_s_at) using the Kaplan–Meier plot applica-tion (ref. 33; http://kmplot.com). Distributions for high and lowcut-off values (Supplementary Fig. S3), P values, HRs, and con-fidence intervals for all survival analyses can be found in Supple-mentary Materials (Supplementary Table S6).

Clinical tumor specimensPrimary breast tumor specimens were acquired from theUCBC

(Athens,GA;n¼8).UCBCpatientswere newly diagnosed (within�1 month), and following informed consent at time of enroll-ment, primary tumor samples were collected at local Athens areahospitals. There were no exclusion criteria for this study cohort,which consisted of a racially diverse patient group having variousmolecular subtypes of breast cancer (Table 1). Tumor grade wasdetermined using WHO guidelines, as well as the Ellis & Elstonsystem of histologic grading. Clinical staging was evaluated usingthe TNM staging system maintained by American Joint Commit-tee on Cancer and Union for International Cancer Controlfollowing the most current National Comprehensive CancerNetwork guidelines at the time of staging.Molecular breast cancersubtypes were determined using IHC staining for estrogen recep-tor, progesterone receptor, and human epidermal growth factorreceptor-2 (HER2) on the surface of the primary breast tumor.

IHCFormalin-fixed paraffin-embedded tumor blocks were

obtained from local Athens-area hospitals through UCBC(Athens, GA). Subsequent slide preparations were conductedthrough the University of Georgia's Histology Core, using stan-dard operating protocols (FFPE blocks used to cut 4 mm sections

Table 1. Case–control cohort baseline characteristics

Characteristic Cases, n (%) n ¼ 266 Controls, n (%) n ¼ 156

Age (years)Mean (�SD) 62.69 (�11.79) 46.10 (�16.34)Median (range) 63 (33-92) 45 (20-79)

EthnicityWhite-American 190 (71.43) 111 (71.15)African-American 76 (28.57) 45 (28.85)

Molecular subtypeLum A 174 (65.41) —

Lum B 25 (9.40) —

HER2þ 37 (13.91) —

Basal-like 25 (9.40) —

HRþ 5 (1.88) —

Clinical stageI 183 (68.80) —

II 62 (23.31) —

III 10 (3.76) —

IV 5 (1.88) —

Other 5 (1.88) —

Not available 1 (0.37) —

DARC/ACKR1 Genotype (rs2814778)AA 157 (73.71) 110 (73.33)AG 23 (10.80) 15 (10.00)GG 33 (15.49) 25 (16.67)

DARC/ACKR1 RBC PhenotypePositive 145 (69.05) 42 (89.36)Intermediate 41 (19.52) 3 (6.38)Negative 24 (11.43) 2 (4.26)

Abbreviations: HR, hormone receptor; LumA, Luminal A; LumB, Luminal B; RBC,red blood cell.

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onto glass slides). DARC/ACKR1 staining was done using a goatanti-human polyclonal antibody (Novus Biologicals, NB100-2421, isotype IgG). CCL2 and CXCL8 staining were both doneusing mouse anti-human mAbs (R&D Systems, MAB2791, iso-type IgG2B and Lifespan Biosciences, LS-B6427, isotype IgG1,respectively).

Statistical analysisStatistical analyses were done using appropriate functions in

JMP (SAS) Pro v. 13.0.0. All Student t tests performed were two-tailed and the appropriate absolute P values are indicated in thefigure legends of each statistical analysis. F values are reportedfor ANOVA analyses and P values are reported for linearregression or paired t test analyses. Numbers of included datapoints and degrees of freedom are also shown for the appro-priate analyses.

ResultsDARC/ACKR1 tumor expression is significantly different acrossrace groups and tumor molecular subtypes

Our previous studies of genomic signatures in breast cancersubtypes indicated an enrichment in immune-related genepathways (34). We identified a pattern of dichotomous varia-tion, measured by first-generation microarray platforms fromGEO (35, 36) in a chemokine receptor that regulates immunecell signaling molecules, the DARC/ACKR1 gene. To quantifythese findings more robustly, we investigated DARC/ACKR1expression differences at a higher resolution, using the TCGA

breast-infiltrating carcinoma RNAseq dataset (n ¼ 838; 167AAs, 671 WAs), and found a broad range and distribution ofDARC/ACKR1 expression across the cohort (Supplementary Fig.S1A and S1B). A statistical response screen across anthropo-morphic and clinical variables suggested that DARC/ACKR1tumor expression was distinct across race and tumor pheno-types (Supplementary Fig. S1C and S1D). Our analysis revealedthat WAs have a nearly equal distribution of DARC/ACKR1tumor expression subgroups, with the highest proportion beingDARC/ACKR1-high (35.3%), and AAs have the highest propor-tion of DARC/ACKR1-low tumors (40.1%; Fig. 1A, top). Theserace-related trends indicate a distinct regulation of DARC/ACKR1 that is likely linked to geographic genetic ancestry,which is closely associated with race.

However, because DARC/ACKR1 expression occurs acrossall organ systems within endothelial cells of postcapillaryvenules (24, 37), we also considered the relative expression ofan endothelial cell–type marker, Von-Wilderbrand Factor (VWF)within the tumor samples.When tumors were stratified byDARC/ACKR1 status and VWF (Fig. 1A, bottom), the differences ofDARC/ACKR1 status between race groups were more evident(P¼ 0.06), just above the threshold for significance. Interestingly,the correlation of DARC/ACKR1 to VWF was very significant(P < 0.0001, Supplementary Fig. S1F), which suggests that asignificant portion of DARC/ACKR1 expression within thesetumor samples might be derived from endothelial tissue. How-ever, even within tumors with low VWF, we still find highexpression of DARC/ACKR1, particularly in WA patients.

Figure 1.

DARC/ACKR1 is significantly associated with VWF, breast cancer molecular subtypes, and pro-inflammatory chemokines in TCGA data. TCGA Breast CancerRNAseq data (n¼ 838) were used to compare (A, top) the distribution of DARC/ACKR1 expression subgroups (high, n¼ 289, pink; medium, n¼ 268, yellow; low,n¼ 281, purple) and race (AA, n¼ 167; WA, n¼ 671). Bottom panel of A same as top but with VWF expression subgroups (VWF Low, n¼ 336; VWFMid, n¼ 309;VWF High, n¼ 193, P¼ 0.06, ANOVA) by race. B, Distribution of DARC/ACKR1 expression subgroups compared with molecular breast cancer subtypes (Basal-like, n¼ 74; HER2þ, n¼ 24; Luminal A, n¼ 196; Luminal B, n¼ 58, P <0.0001). Bottom panel of B same as top but broken down by race. C, Heatmap (UCSC XenaBrowser) of TCGA breast-invasive carcinoma RNAseq gene expression data (IlluminaHiSeq) shows cytokine expression after creating dichotomized DARC/ACKR1-positive (red) and -negative (blue) subgroups. Gene expression was assessed in 399 breast tumors against a panel of 67 genes associated with knowncytokines (blue, low expression; red, high expression). Welch t test was performed on CCL2 (P¼ 0.0000, t¼ 10.28) and CXCL8 (P¼ 0.0003, t¼�3.644).D, DARC/ACKR1 high and low categories compared with CCL2 (left, Student t test, P < 0.0001, DF¼ 438.05) and CXCL8 (right, Student t test, P < 0.0001,DF¼ 376.25) gene expression (mean� SEM).





In the context of current standards of breast tumor molecularphenotypes, we also found that DARC/ACKR1 tumor status (e.g.,DARC/ACKR1-high vs. DARC/ACKR1-low) was significantly dis-tinct across the PAM50 (36) subtype categories (P < 0.0001), asdefined by RNAseq gene expression profiles (Fig. 1B, top). Spe-cifically, nearly 40%of the PAM50 Luminal A subtypes areDARC/ACKR1-high tumors, which is the largest proportion of DARC/ACKR1-high tumors across all subtypes. Contrarily, the highestproportion of DARC/ACKR1-low tumors is within the Basal-likesubtype, making up over 60% of this category. When the PAM50subtypes are also stratified for (self-reported) race (Fig. 1B, bot-tom), weobserved clear distinctions in the distributions ofDARC/ACKR1 tumor status between AA and WA, although these differ-ences did not pass thresholds for statistical significance.

To investigate whether the status of DARC/ACKR1 tumorexpression is connected to genes that function in DARC/ACKR1-related chemokine signaling pathways, we investigatedover 60 chemokine genes (Supplementary Table S1) and foundthat there were significant differences in chemokine gene expres-sion, correlated with DARC/ACKR1 status (Fig. 1C; Supplemen-tary Table S2). Two of these genes, CCL2 and CXCL8, arehigh-affinity DARC/ACKR1-binding targets (38, 39) and havesignificant expression in breast tumors (Supplementary Fig.S4A) correlated with DARC/ACKR1 expression (SupplementaryFig. S4C and S4D). When compared across DARC/ACKR1 tumortypes,we found thatDARC/ACKR1-high tumors have significantlycorrelated high levels of CCL2 (P < 0.0001), but significantlyanticorrelated lower levels of CXCL8 in primary tumors(P < 0.0001; Fig. 1D).

Circulating levels of DARC/ACKR1-related chemokines, CCL2and CXCL8, are associated with disease status, Duffy-nullphenotype, self-reported race, and African ancestry–specificalleles of DARC/ACKR1

We conducted a Luminex analysis in an independent breastcancer case–control cohort (case, n ¼ 266, controls, n ¼ 156) toidentify associations among DARC/ACKR1-related chemokinesand histopathologic variables in our cohort. When comparingrace between cases and controls overall, we identified a significantdifference in circulating CCL2 levels between AA cases and WAcases, where AA had significantly lower CCL2 than WA (P ¼0.0031, Fig. 2A, top). We did not observe a significant differencebetween cases and controls in AA, but CCL2 levels were decreasedin AA cases only, a trend we did not observe in WA. We alsoobserved a significant increase in circulating CXCL8 levels in AAcases (P ¼ 0.0013, Fig. 2A, bottom) and WA cases (P <0.0001, Fig. 2A, bottom) compared with their respective controls.These lower circulating CCL2 protein expression levels in AAscompared with WAs is a trend that indicates a potential race-specific circulatory inflammatory response; however, this sametrend was not observed in the tumor gene expression data fromTCGA (Supplementary Fig. S4B).

To determine the distribution and impact of the Duffy-nullphenotype on cancer-related inflammatory response in ourcohort, we tested our cases and controls for the erythrocyte-silentphenotype (Fig. 2B), as well as the Duffy-null gene mutation(rs2814778). As expected, we found both a higher frequency ofthe DARC/ACKR1es/Duffy-null blood group and the Duffy-null/ACKR1es allele (rs2814778) frequency in AAs (Fig. 2C). These

Figure 2.

DARC/ACKR1 phenotype and genotype associated with race and proinflammatory chemokines in case–control cohort (n¼ 422). A, Case–control comparison ofcirculating CCL2 (top) and CXCL8 (bottom) levels by race (Student t test, P¼ 0.0031, DF¼ 182.09). B, Representative images of DARC/ACKR1 phenotyping onRBCs; BF, bright field; DARC/ACKR1, green; plasmamembrane control, red; scale, 50 mm. C, Distribution of DARC/ACKR1 phenotype [top, positive¼ purple;intermediate¼ pink; negative¼ yellow, n (AA)¼ 71, n (WA)¼ 186], and genotype [bottom, rs2814778; AA¼ purple, homozygous reference; AG¼ pink,heterozygous; GG¼ yellow, homozygous alternate, n (AA)¼ 101, n (WA)¼ 262] compared with race. D, DARC/ACKR1 phenotype on RBCs compared withcirculating CCL2 (left, Student t test, P¼ 0.0436, DF¼ 42.88) and CXCL8 levels (right); Inter.¼ intermediate. E, Same asD, but with DARC/ACKR1 genotype(rs2814778; CCL2, left, ANOVA, F¼ 0.0002, DF¼ 212, Student t test, P <0.0001, DF¼ 137.31, Student t test, P¼ 0.0176, DF¼ 27.47, CXCL8, right).

Jenkins et al.




distributions were in concordance with published global andpopulation-specific allele frequencies (Supplementary TableS4). We then compared circulating levels of CCL2 and CXCL8with the DARC/ACKR1 RBC phenotype and Duffy-null genotypefor case and control groups (Fig. 2D and E). For phenotype, wefound significant differences in CCL2 levels between those withpositive DARC/ACKR1 erythrocyte expression and negativeexpression among the newly diagnosed breast cancer cases only(P¼ 0.0436; Fig. 2D, left). When considering genotype, we foundsignificant differences in CCL2 levels across all newly diagnosedcases (P ¼ 0.0002; Fig. 2E, left). We also observed individualassociations between those that are homozygous for the referenceallele (AA) and homozygous for the alternate Duffy-null allele(GG, P < 0.0001; Fig 2E, left), and those that are heterozygous andhomozygous for the Duffy-null allele (P ¼ 0.0176; Fig 2E, left)in newly diagnosed cases. This correlation was similar whenthe phenotype groups were broken down by race (SupplementaryFig. S5). Interestingly, the significant differences in CCL2-

circulating levels increased among the DARC/ACKR1 allele car-riers (Fig. 2E) indicating that the genetic allele is a more robustindicator of these circulating chemokine levels than the RBCphenotype, in newly diagnosed patients.

DARC/ACKR1 tumor gene expression is associatedwith distincttumor-associated leukocyte (TAL) gene expression profiles

Because the chemokines under regulation of DARC/ACKR1direct the infiltration of immune cells during inflammatoryresponses, we investigated the differences in immune cellresponses, relative to DARC/ACKR1 tumor expression. UsingTCGA RNAseq data, we employed a cell-type deconvolutionalgorithm, CIBERSORT, and determined that overall leukocyteinfiltration (total TAL score) is greater in DARC/ACKR1-hightumors (Fig. 3A; ref. 31). These counts were directly correlatedwith quantified DARC/ACKR1 gene expression (SupplementaryFig. S6A), suggesting that DARC/ACKR1 could be a marker thatpredicts the level of tumor immunogenicity. The increase of total

Figure 3.

DARC/ACKR1 expression levels are positively and significantly associated with TAL abundance in breast tumors. Using CIBERSORT absolute mode, the absolutescore totals the quantitative abundance of each individual leukocyte population among TCGA breast primary cases. Those cases with significant CIBERSORTresults (P <0.05) were included in this analysis (n¼ 472). A, The total TAL absolute score reported is a total of abundance scores across all 22 TAL populations.By plotting total TAL absolute score by DARC/ACKR1 expression status, we see significant increases in TAL abundance as DARC/ACKR1 expression is increasingbetween low, medium, and high categories. Looking at individual TAL populations, B shows the 12 leukocyte populations that are significantly different betweenDARC/ACKR1 expression levels. Student t test was performed comparing each DARC/ACKR1 expression group, and the P values for the associations arerepresented by the heatmap in C. Highly significant associations, dark red; less significant associations, dark blue.





immune cell infiltrates was also correlated with increasing CCL2,but to a lesser extent (Supplementary Fig. S6B), indicating thatCCL2 has some influence on, but is not sufficient for, the totalobserved immune cell profiles.

We found distinctions in both the proportions and combina-tions of specific infiltrating immune cell subtypes of DARC/ACKR1-high tumors compared with DARC/ACKR1-low tumors.Specifically, there is an increase in numbers of B cells, T cells (CD8andCD4), T-regulatory cells, and activatedmacrophages (M1 andM2; Fig. 3B andC). Conversely,DARC/ACKR1-low tumors appearto lack dendritic and B (memory) cells. Differences in the DARC/ACKR1-associated immune cell–type profiles are also presentamong race groups (P ¼ 0.08). Specifically, AA patients with theDARC/ACKR1-low tumor subtype have more infiltrating M0macrophages and fewer B cells compared with WA patients(Supplementary Fig. S6C).

HigherDARC/ACKR1 tumor gene expression is associated withlonger survival

To determine the clinical significance of DARC/ACKR1 geneexpression in breast tumors, we investigated publicly availabledatasets for associations of DARC/ACKR1 tumor gene expressionwith survival (cohorts are summarized in Supplementary TableS6). We found that when patients have higher DARC/ACKR1tumor expression, they have significantly longer OS and RFS(Fig. 3A and B; OS P < 2.2 � 10�6, n ¼ 1,402; RFS P < 1 �10�16, n ¼ 3,951). This survival association remained significantfor RFS across all molecular subtypes (including; Luminal A,Luminal B, Basal-like), with one exception, HER2þ (Fig. 4A–E).

Similarly, the <5-year survival outcome in TCGA is also linked toDARC/ACKR1 tumor expression (Supplementary Fig. S1 G). Theimpact of CCL2 (Fig. 4G–L) and CXCL8 (Fig. 4M–R) can also beseen within certain molecular subtypes of breast cancer, but oftento a lesser degree thanDARC/ACKR1 associations. In all subtypes,it is clear that inflammatory markers related to DARC/ACKR1tumor expression have a significant impact on survival. Specifi-cally, CCL2 has the most significant survival impact on basal-likeand HER2þ cases (Fig. 4K and L) where higher CCL2 is associatedwith longer survival. Contrarily, CXCL8 also has a significantimpact in basal-like cases (Fig 4Q), but in the opposite direction,where low expression of CXCL8 leads to longer survival. Thisopposing trend of CCL2 and CXCL8 is directly associated withDARC/ACKR1 status, as shown previously in Fig. 1D.

In tumor epithelial cells, DARC/ACKR1 is correlated withhigher CCL2 and lower CXCL8 by IHC data

DARC/ACKR1 has been shown to be expressed on endothelialpostcapillary venules, which are present in normal breast tissue(Supplementary Fig. S7A) and tumors as well. However, ourpreliminary data in breast cell lines indicated DARC/ACKR1 wasexpressed in tumor cells (Supplementary Fig. S7B). To confirm thegenomic and cell line data, we conducted IHCona small cohort ofprimary tumor specimens (n ¼ 8) to validate the spatial expres-sion of DARC/ACKR1 in the tumor microenvironment. Ourinitial results indicate that when DARC/ACKR1 is expressed, itwas not limited to endothelial cells, but was uniformly expressedwithin the tumor-specific epithelial cells. This finding validatesthe VWF-associatedDARC expression trends shown in Fig. 1A and

Figure 4.

High DARC/ACKR1 tumor expression is associated with significantly higher survival in breast cancer. Kaplan–Meier survival curves (www.kmplot.com) of patientswith breast cancer dichotomized into DARC/ACKR1 (208335_s_at, top row), CCL2 (216598_s_at, middle row), and CXCL8 (211506_s_at, bottom row) low(black) and high (red) categories showing overall survival (A, G, M) and relapse-free survival (B–F, H–L, N–R) by all molecular subtypes and four individualsubtypes (Lum A, Luminal A; Lum B, Luminal B; Basal-like, and HER2þ, Human Epidermal Growth Factor Receptor 2). Beeswarm distribution plots, HRs, andconfidence intervals can be found in Supplementary Materials.

Jenkins et al.



www.kmplot.com


Supplementary Fig. S1F. Specifically, our IHC subset suggests thatthe DARC/ACKR1 gene product is also expressed over the fullspectrum of scoring (0–4) in our cohort (Fig. 5A), similar to theRNAseq distributions (Supplementary Fig. S1A and S1B). Despitethe small size of the pilot IHC cohort (n ¼ 8), we observed asimilar trend as the RNAseq findings, where tumors with highDARC/ACKR1 IHC scores showed correlated high CCL2 expres-sion and low CXCL8 expression (Fig. 5A).

Anticipating that the role of DARC/ACKR1 on epithelial cellswould be similar to that on endothelial cells, we investigatedwhether circulating chemokine levelswere associatedwithDARC/ACKR1 tumor scores. We integrated the Luminex chemokineassays from our clinical cohort with a pilot IHC study with asubset of cases and found a significant positive correlationbetween DARC/ACKR1 tumor scores and circulating levels ofCCL2 (P ¼ 0.0061) and CXCL8 (P ¼ 0.0291; Fig. 5B; Supple-mentary Table S3). This findingwas also alignedwith our RNAseqfindings for CCL2, CXCL8, and DARC/ACKR1 levels.

DiscussionDistinct tumor immune responses among ancestry groups withcancer disparities

Disparities in cancer outcomes among race groups paralleldistributions of tumor phenotypes, including distinct signaturesof tumor-specific gene expression signatures. These findings haveshifted the lens of clinical disparities research to now encompassmolecular investigations of tumor phenotypes that drive poorprognoses, which can be now be linked to genetic ancestry,particularly West African Ancestry.

Work from our group and others uncovers molecular mechan-isms of cancer that parallels the mortality and tumor phenotypeincidence among groups of African and AA women (12). Geno-mic studies conducted in large AA and/or multi-ethnic cohortshave begun to utilize modified genomic tools in search of pop-ulation-specific risk alleles. SNVs, structural, chromosomal, andepigenetic variations are being discovered and associated with a

still theoretical genetic predisposition for the distinct tumorbiology observed among women of West African descent (40–43). While more risk alleles are being detected through increaseddiversity ofmulti-ethnic genome-wide association study (GWAS),the specific risk alleles transmitted into non-white populationsthrough African genetic ancestry have not yet been definitivelyshown (44). Our current study deepens our understanding of arepeated finding among gene expression studies, which describedifferences in disease biology rather than risk of disease incidence/predisposition among race groups.Multiple groups have reportedAA (race-specific) expression profiles that implicate immuneresponse pathways in a distinct category of disease progres-sion (45–47), but again without definitive links to African geneticancestry.

In this report, we have shown that a West African–specificallele of DARC/ACKR1, a gene that regulates immune andinflammatory response, is strongly associated with both theepithelial expression and circulating levels of CCL2 and CXCL8,which are proinflammatory chemokines previously shown to berelated to cancer progression. We have thus implicated theepithelial tumor expression of DARC/ACKR1 to be a key mod-ulating factor of chemokines in tumors, putatively confirmed insmall IHC study within our clinical cohort. These findingssuggest that DARC/ACKR1 has a dual role in controlling che-mokine gradients that determine tumor immune cell response,specifically by regulating both the levels of chemokines intumors and in circulation. Interestingly, tumor expression ofCXCL8 is negatively correlated with DARC/ACKR1 tumor expres-sion while circulating levels of CXCL8 are positively correlatedwith DARC/ACKR1 tumor expression. These opposing trends arerecapitulated in our survival analyses where high levels ofDARC/ACKR1 in tumors and/or low levels of CXCL8 yield longersurvival. We hypothesize this is an unmasking of a distinctionin DARC/ACKR1 control of CXCL8 compared with CCL2 thatmay be related to DARC/ACKR1 tumor activity involving clear-ance of CXCL8 from the tumor and/or DARC/ACKR1 isoformfunctionality, which we are currently investigating.

Figure 5.

DARC/ACKR1 may correlate with proinflammatory chemokines in breast tumor tissue. A, Primary breast tumors stained for DARC/ACKR1 (dark pink), CCL2 (lightpink), and CXCL8 (brown) by IHC and scored on a numerical system of 0–4, scale bar¼ 200 mm. B, Linear correlation between DARC/ACKR1 IHC scores (graybars) and circulating CCL2 (straight line) and CXCL8 (dotted line) concentrations from Luminex assay (n¼ 8).





Previous studies have already shown that the spatial expressionof DARC/ACKR1 is governed, at least in part, by the Duffy-nullallele (rs2814778). Given that AA cohorts have over 70% allelefrequency of this allele (48, 49), we anticipate that most AA'swould carry the allele and therefore have distinct regulation of thegene in certain tissues. On the basis of our findings, this allelewould predispose patients with breast cancer, who are Duffy-nullcarriers, to having a specific tumor phenotype, defined by thetumor immune cell landscape, creating a tumor immune responsethat is directed by the DARC/ACKR1 expression in tumors. Ourcase–control cohort indicates that the Duffy-null genotypes are astronger indicator of circulating chemokines, compared withcorrelations that use self-identified race. The differences in che-mokine correlations among race groups, compared with strictheterozygote/homozygote allele groups, corresponds to whatwould be expected within an admixed population where allelesrelated to genetic ancestry are convolutedwithin the proxy of race,where a portion of the individuals that may self-identify withAfrican ancestry are harboring a non-African allele. Therefore, theassociations of DARC/ACKR1 and chemokine levels in breastcancer cases can be rationally interpreted as ancestry-associated.

Whilewehave found this association inour study,webelieve thisallele has not been identified inmulti-ethnicGWAS that investigatecancer risk among race groups (22, 50) because this genewouldnotimpact tumor etiology, but rather disease progression, and there-fore does not qualify as a "disease (onset) risk" allele. However,when prevention fails, survival becomes imperative. This allele isassociated strongly with both OS and RFS across all breast cancersubtypes and is linked to race through genetic ancestry. Therefore,this important work begins to unravel howAfrican ancestry directlyimpacts the tumor biology and microenvironment of tumor pro-gression, even systemically. We suspect that the trends of DARC/ACKR1 tumor expression were not quite significant in the TCGAdata simply due to lack of true African ancestry measurements inlieu of self-identified race proxies. The genetic admixture of Euro-peangenetic inheritancewithin theAAgrouphas likely confoundedDARC/ACKR1 tumor expression race associations. We will seek tofollow-up with genetic ancestry estimates in these cases in asubsequent validation cohort.

The cancer-related mechanisms that derive aggressive tumorphenotypes, such as TNBC, have yet to provide clear and action-able markers for therapeutic decisions in clinic. Studies that seekto identify the factors that lead to treatment-refractory tumorconditions have often implicated the tumor microenvironment(TME; ref. 51). A significant component of the TME includesimmune cells that are either associatedwith or infiltrating into thetumor and tumor-associated stroma (21). The assortment ofimmune cells that respond to tumor formation can determinethe course of tumor progression (52). Tumor immunology stud-ies suggest that TALs can lead to either good or bad prognoses,such as with higher proportions of B cells versus T cells, respec-tively (53–57). While our ability to discern immune cell types inthe tumor space has great prognostic value, it is a tedious task toquantify each TAL cell type to establish adverse versus favorableimmuneprofiles and is not feasible on a case-by-case basis inmostclinics. Our discovery of a single genetic marker that coulddefinitively regulate the course of immune response in tumorsand therefore could forecast TAL profiles would be an invaluablebiomarker to identify patients suitable for immunotherapies. Inour report, we have introduced such a potential biomarker,showing that tumor expressionof an atypical chemokine receptor,

DARC/ACKR1, is associated with immune response. This associ-ation is aligned with the gene's extensively defined regulation ofchemokine levels in circulation, which may now also include theregulation of these levels in tumors as well. This suggests thatDARC/ACKR1 can modulate the chemoattractants in patientswith breast cancer, ultimately directing the actions of immunecells within the tumors.

With further investigations that characterize and quantifyDARC/ACKR1-dependent TAL infiltration, in the context of Afri-can ancestry, we will develop a systematic approach to determinewhether DARC/ACKR1 status may predict immune responseprofiles in breast tumors and ultimately predict immunotherapytreatment responses. This current study has established the fea-sibility of DARC/ACKR1 as a marker to define a novel tumorsubtyping that defines its immune status. A subset of our cohortstudy, reported here, indicates the feasibility of integrating single-marker tumor IHCwith amulti-marker peripheral blood assay. Incombination, our data have shown that differences in tumorimmune cell profiles, which are relevant to race, are associatedwith the tumor expression of DARC/ACKR1. Specifically, theAfrican-specific Duffy-null variant of DARC/ACKR1, which isalready associated with the gene's spatial expression, determinesa unique biological scenario for distinct TAL infiltrates. Thislandmark finding has high relevance in cancer management, asa functional regulator of proinflammatory and chemotactic cyto-kines that can specify an infiltrating tumor immune cell–typeprofile and specify differences between patients of distinct ances-try. This further implicates DARC/ACKR1 in tumor phenotypedistinctions that could significantly drive treatment outcomes thatlead to cancer mortality disparities. Indeed, DARC/ACKR1 mayalso begin to unravel the racial disparities of treatment outcomeswithin tumor phenotype categories that have effective targetedtherapies and the overall outcomes in TNBC (57).

As with any epidemiologic study, limitations of our studyinclude confounding factors within cohorts that are inseparablefrom variables of interest. Specifically, differences in DARC/ACKR1 gene expression between race groups could also be afunction of a well-documented bias in occurrence of specificmolecular tumor phenotypes across these populations (58). Forinstance, women of African descent have a higher relative inci-dence of TNBC, and we see this same trend in our cohort andincluded supplementary analysis of race and subtypes in Supple-mentary Data. Interestingly, the Duffy-null allele has previouslybeen implicated in other immune-related diseases, such as neu-tropenia (59), and therefore the distinct chemokine levels weobserve in AA patients, associated with the mutation, now impli-cates a novel role of DARC/ACKR1 in a breast cancer disparitiescontext. These correlations in chemokine levels were found pri-marily in newly diagnosed cases, suggesting that DARC/ACKR1plays a similar role in tumors as what has been shown inendothelial cells—facilitating the entry of tumor-associated che-mokines into circulation (i.e., transcytosis; ref. 26)—and so itmaybe regulating increase in circulating chemokine levels by facili-tating chemokine transfer from epithelial tumor cells. Therefore,despite our limitations, these compelling findings indicate thatthe African-specific allele of DARC/ACKR1 modulates cancer-driven circulating chemokine levels and therefore may regulatethe infiltration of tumor-associated immune cells we observed intumors expressing DARC/ACKR1 at high levels compared withthe lower numbers of tumor-associated immune cells whenDARC/ACKR1 is expressed at low levels, if at all. Our ongoing

Jenkins et al.




investigations in larger cohorts with higher numbers of non-whitecases and better representation of all intrinsic tumor subtypes canfurther solidify the race/ancestry findingswe have uncovered here.

Disclosure of Potential Conflicts of InterestC. Yates is a consultant and has ownership interest at Riptide Biosciences. L.

Newman is an attending physician at Henry Ford Health System. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: B.D. Jenkins, R. Hire, H. Ali, M. Monteil, M.B. DavisDevelopment ofmethodology:B.D. Jenkins, R.Hire, B. Bennett, E.W.Howerth,M.B. DavisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.D. Jenkins, R. Hire, A. Brown, I. Brown,E.W. Howerth, M. Egan, J. Hodgson, D. Chitale, D. Nathanson,P. Nikolinakos, L. Newman, M.B. DavisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):B.D. Jenkins, R.N.Martini, R.Hire, I. Brown, R.Kittles,L. Newman, M. Monteil, M.B. DavisWriting, review, and/or revision of themanuscript: B.D. Jenkins, R.N.Martini,R. Hire, E.W. Howerth, C. Yates, R. Kittles, D. Chitale, H. Ali, P. Nikolinakos,L. Newman, M. Monteil, M.B. DavisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B.D. Jenkins, B. Bennett, L. Newman,M.B. Davis

Study supervision: P. Nikolinakos, M.B. DavisOther (contributed breast cancer material from his surgical practice):D. Nathanson

AcknowledgmentsWe are eternally grateful for the donors and volunteers who have sup-

ported our research efforts through participation and donating their bloodor tissue for use as cohort samples. These brave women include the parti-cipants of the "Be the Research" program at UGA, TCGA patients, KomenBiospecimen Tissue Bank, and the Henry Ford International Breast Registry.B.D. Jenkins is a Howard Hughes Medical Institute Gilliam Fellow. Specialthanks to Ben Rybicki and Nancy Manley for helpful discussions on datainterpretations, and study scope and impact. This work was supported bygrants: R21-CA210237-03 (NIH/NCI; to M.B. Davis), U54-MD007585-26(NIH/RCMI; to C. Yates), U54 CA118623 (NIH/NCI; to C. Yates), (NIH/NCI) 1 R21 CA188799-01 (to C. Yates), and SAC160072 (Komen; to L.Newman).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 27, 2018; revised November 11, 2018; accepted January 4,2019; published first April 3, 2019.

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Jenkins et al.



2019;28:690-700. Cancer Epidemiol Biomarkers Prev Brittany D. Jenkins, Rachel N. Martini, Rupali Hire, et al. Tumor-Infiltrating Immune Cells, and African AncestryIs Associated with Survival, Circulating Chemokines,

) in Breast TumorsDARC/ACKR1Atypical Chemokine Receptor 1 (

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