Translational Cancer Mechanisms and Therapy

IFNL4-DG Allele Is Associated with an InterferonSignature in Tumors and Survival of African-American Men with Prostate CancerWei Tang1, Tiffany A.Wallace1, Ming Yi2, Cristina Magi-Galluzzi3,Tiffany H. Dorsey1, Olusegun O. Onabajo4, Adeola Obajemu4, Symone V. Jordan1,Christopher A. Loffredo5, Robert M. Stephens2, Robert H. Silverman6,George R. Stark6, Eric A. Klein7, Ludmila Prokunina-Olsson4, and Stefan Ambs1

Abstract

Purpose: Men of African ancestry experience an exces-sive prostate cancer mortality that could be related toan aggressive tumor biology. We previously described animmune-inflammation signature in prostate tumors ofAfrican-American (AA) patients. Here, we further decon-structed this signature and investigated its relationshipswith tumor biology, survival, and a common germlinevariant in the IFNl4 (IFNL4) gene.

Experimental Design: We analyzed gene expression inprostate tissue datasets and performed genotype and sur-vival analyses. We also overexpressed IFNL4 in humanprostate cancer cells.

Results: We found that a distinct interferon (IFN) signa-ture that is analogous to the previously described "IFN-related DNA damage resistance signature" (IRDS) occurs inprostate tumors. Evaluation of two independent patientcohorts revealed that IRDS is detected about twice as often

in prostate tumors of AA than European-American men.Furthermore, analysis in TCGA showed an association ofincreased IRDS in prostate tumors with decreased disease-free survival. To explain these observations, we assessedwhether IRDS is associated with an IFNL4 germline variant(rs368234815-DG) that controls production of IFNl4, atype III IFN, and is most common in individuals of Africanancestry. We show that the IFNL4 rs368234815-DG allelewas significantly associated with IRDS in prostate tumorsand overall survival of AA patients. Moreover, IFNL4 over-expression induced IRDS in three human prostate cancercell lines.

Conclusions: Our study links a germline variant that con-trols production of IFNl4 to the occurrence of a clinicallyrelevant IFN signature in prostate tumors that may predom-inantly affect men of African ancestry. Clin Cancer Res; 24(21);5471–81. �2018 AACR.

IntroductionProstate cancer incidence and mortality rates are highest

among men of African ancestry (1–3). Environmental expo-sures and ancestry-specific factors may influence prostate can-cer biology and cause a more aggressive disease in these men(4–11). We and others described an immune signature that is

prevalent in prostate tumors of African-American (AA) patientsand hypothesized that this signature affects tumor biology(5, 12–15). Here, we further investigated this immune signa-ture and discovered an IFN signature in tumors of AA patientsthat is analogous to the previously described IFN-relatedDNA damage resistance signature, also termed IRDS (16). IRDSincludes 49 IFN-stimulated genes (ISG) that are inducedthrough activation of the JAK–STAT pathway (17). IRDS wasinitially identified because of its effects leading to resistanceto resistance to ionizing radiation (17). It can be induced bypersistent activation of the JAK–STAT pathway by variousexogenous and endogenous stimuli that generate an IFNresponse (18, 19).

All IFNs (type I, type II, and type III) induce sets of ISGs thatare overlapping but also distinct. However, among the humanIFNs, the expression of the recently discovered IFNl4, a type IIIIFN, is uniquely controlled by genetics. Only carriers of theDG allele for the germline variant rs368234815-DG/TT inthe IFNL4 gene can produce this IFN (20). This allele is mostcommon in individual of African ancestry (up to 80% allelefrequency), although it is less common in Europeans (�30%)and Asians (less than 10%; ref. 20). Moreover, carriers of IFNL4rs368234815-DG have an impaired ability to clear certain viralinfections, such as hepatitis C virus (HCV), spontaneously or

1Laboratory of Human Carcinogenesis, Center for Cancer Research (CCR), NCI,NIH, Bethesda, Maryland. 2Cancer Research Technology Program, Leidos Bio-medical Research, Inc., Frederick National Laboratory for Cancer Research,Frederick, Maryland. 3Department of Pathology, Cleveland Clinic, Cleveland,Ohio. 4Laboratory of Translational Genomics, Division of Cancer Epidemiologyand Genetics, NCI, NIH, Bethesda, Maryland. 5Cancer Prevention and ControlProgram, Lombardi Comprehensive Cancer Center, Georgetown UniversityMedical Center, Washington, DC. 6Department of Cancer Biology, LernerResearch Institute, Cleveland Clinic, Cleveland, Ohio. 7Glickman Urological andKidney Institute, Cleveland Clinic, Cleveland, Ohio.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Stefan Ambs, NCI, Building 37, Room 3050B, MSCConvent Drive 4258, Bethesda, MD 20892. Phone: 240-760-6836;Fax: 240-541-4496; E-mail: ambss@mail.nih.gov

doi: 10.1158/1078-0432.CCR-18-1060

�2018 American Association for Cancer Research.

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after treatment (20). One of the mechanisms by which IFNl4renders cells refractory to an antiviral response is the inductionof a persistent gene signature that resembles IRDS (20, 21).Thus, we hypothesized that because IFNl4 is produced morecommonly in men of African ancestry, it might explain theincreased occurrence of IRDS in their tumors, and be of clinicalimportance. Accordingly, we examined whether IFNL4rs368234815-DG is associated with the development of IRDSin prostate tumors and disease outcomes. Consistent with ourhypothesis, we found that IFNL4 rs368234815-DG is signifi-cantly associated with both the presence of IRDS and increasedall-cause mortality among AA prostatectomy patients.

Materials and MethodsStudy design, patient data, and cell lines

To compare gene expression profiles from prostate tumors ofAA and European-American (EA) patients, we analyzed geneexpression data from 2 patient cohorts that were previouslydescribed in detail by us (12) and others (22). These two datasetsare publicly available (GSE6956 and GSE21032) and consist ofmicroarray data for primary prostate tumors from 33 AA and36 EA men (12) and 24 AA (non-Hispanic) and 98 EA men (22).These men were previously untreated prostatectomy patientswith exception of 16 patients in the Taylor and colleagues' cohortwho received neoadjuvant hormone therapy or chemotherapy.We examined the prevalence of two previously reportedIFN-related gene signatures in the tumors, IRDS (16) and IFN-regulated genes (IRG; ref. 23). As described in the publications,IRDS and IRG include 49 and 42 ISGs, respectively. To assess theassociation of IRDS with disease-free survival, we evaluated thepublicly available TCGA prostate cancer data accessiblethrough the Cancer Genomics Data Server (CGDS, at http://www.cbioportal.org/public-portal) and hosted by the Computa-tional Biology Center at Memorial-Sloan-Kettering Cancer Centerthrough cBioPortal for Cancer Genomics. In this large dataset,recurrence-free survival was available for 491 patients who aremainly EAmen (self-reported race/ethnicity: 270 white, 43 black,6 Asian, others unknown). To examine the association betweenIFNL4 rs368234815-DG allele and IRDS in prostate tumors,we genotyped rs368234815-DG in available genomic DNA from

44 frozen tumors in the Wallace and colleagues' cohort (12).We also assessed genome-wide gene expression using a linearregression model in relation to 0, 1, and 2 copies of the DG alleleand applying an additive model. The Wallace and colleaguesstudy has been approved by Institutional Review Boards, asdescribed previously (12). To assess the association of IFNL4rs368234815-DG with disease recurrence and overall mortality,we isolated genomic DNA from tumor-adjacent, noncancerousprostate tissues that were obtained from 197 AA patients withprostate cancer after a prostatectomy at the Cleveland Clinic(1987–2012). According to follow-up through 2014, 92 patientshad a PSA-defined prostate cancer recurrence, and 29 died ofvarious causes (Supplementary Table S1). Clinical informationincluding age at diagnosis, tumor stage, grade (Gleason score),vital status follow-up, and cause of death was available for thesesubjects. Collection of tissues and patient information wasreviewed and approved by the Cleveland Clinic IRB under pro-tocol CCF IRB 313-773. Our research followed the ethical guide-lines set by the Declaration of Helsinki, and informed consentwas obtained from all patients in the study.

Human prostate cancer cell lines 22Rv1 and PC-3 (from EAdonors) and MDA-PCa-2b (from an AA donor) were obtainedfrom the ATCC and have been regularly authenticated using ashort tandem repeat analysis with GenePrint10 and tested forMycoplasma contamination.

IRDS in prostate tumors and associations with IFNL4-DGand disease recurrence

The mRNA expression data for Wallace and colleagues (12)were available in-house, although the data for Taylor and collea-gues (22)were downloaded asnormalized log2 data from the cBioCancer Genomics Portal (http://cbio.mskcc.org/cancergenomics/prostate/data/). Normalized expression data were subjected to apathway-level comparative analysis using the Sample-LevelEnrichment-Based Pathway Ranking (SLEPR) method (24).SLEPR can be used to quantitatively evaluate gene set-levelexpression patterns at the sample level (e.g., relative expressionof a gene signature in a tumor). Pathway-level enrichment assess-ment using SLEPR was applied to Gene Ontology annotations(GO, http://www.geneontology.org) and to the two overlappingexpression signatures, IRDS and IRG. The IRDS and IRG gene listswere obtained from the two publications that described thesesignatures (16, 23). Supplementary Table S2 shows the Affymetrixprobe set composition of IRDS for the GeneChip HG-U133A 2.0arrays. Evaluation of sample-level upregulated genes in a pathwayor predefined gene signature was performed using the one-sidedMADe method (see SLEPR). Computation of pathway-levelenrichment scores for each sample-level differentiated gene wasperformed on the basis of 2 � 2 contingency tables using theFisher exact test. To determine the statistical significance ofenrichment scores at the pathway/gene signature level for classcomparison (e.g., AA vs. EA patients), a P value was calculatedfrom 1,000 permutations. FDR Q-values were computed frompermutated data. Heatmaps to visualize enrichment scores forupregulated genes in a pathway/gene signature at the sample level(e.g., in an AA tumor) were generated using gradients of red color,which indicate the enrichment level [�log (permutated P value)].All procedures for SLEPR and visualization of findings in heat-maps are part of the WPS software (24). For the analysis of theassociation between tumor IRDS and IFNL4 rs368234815-DG,enrichment scores for IRDS were dichotomized and tumors with

Translational Relevance

Tumor IFN signaling has recently been shown to modulateresponse and resistance to immune checkpoint blockade.Here, we describe a distinct and biologically relevant IFNsignature in prostate tumors that has a high prevalence inAfrican-American (AA) patients. This signature, known as"IFN-related DNA damage resistance signature" (IRDS), pre-dicts decreased disease-free survival. Moreover, we link itsoccurrence to a germline variant allele, rs368234815-DG,within the IFNl4 (IFNL4) gene. This IFNL4 allele controlsproductionof a type III IFN, IFNl4, and is a knownpredictor ofdecreased viral clearance. Together, these observations indi-cate that IRDS and IFNL4 rs368234815-DG may have a func-tion in the tumor biology and survival of AA patients, andinfluence immune therapy outcomes, which should be exam-ined in future studies.

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an enrichment score of zero (no significant enrichment abovebackground) were defined as IRDS-negative (n ¼ 25), whereasthe other tumors were defined as IRDS-positive (n ¼ 19).

The relationship between IRDS in prostate tumors with diseaserecurrence was evaluated in RNA sequencing (RNA-seq) data forthe TCGA prostate cancer cohort of 491 patients. We extracted theIRDS expression profile from the TCGARNA-seq data, leading to a45-gene signature with all genes beingmeasurable expressed, andapplied the consensus clustering method to identify distincttumor clusters with differential IRDS expression in the dataset.This was done using the Bioconductor ConsensusClusterPluspackage (https://bioconductor.org/packages/release/bioc/html/ConsensusClusterPlus.html). This method provides a quantita-tive assessment for determining the number of possible clusterswithin the dataset. Initially, we tested 2 to 8 cluster groups. Finalsubgroups were defined according to their cumulative distribu-tion functions (CDF) and the Delta area under the CDF curve,yielding three distinct clusters that represented the most robustclustering inour data. Then,we calculated the IRDS score basedonIRDS expression values and assigned those to the three clusters,yielding robust differences between the clusters (low, medium,and high). We then accessed the association of these three IRDSexpression groups with disease recurrence using Cox regressionmodeling and visualized findings with a Kaplan–Meier plot.

IFNL4 rs368234815-DG genotypingGenomic DNA was isolated from frozen tissues using the

DNeasy Blood and Tissue Kit, (QIAGEN). To isolate DNA fromformalin-fixed, paraffin-embedded (FFPE) tissues, 5-mm sec-tions were deparaffinized and DNA was extracted using theBiOstic FFPE Tissue DNA Isolation Kit (MO BIO Laboratories).DNA quantity and quality were determined by NanoDrop1,000 (Thermo Fisher Scientific) and 10 ng of DNA was usedfor genotyping. A previously described TaqMan assay forrs368234815 (20) was purchased from Life Technologies andused on the ABI 7900 (Applied Biosystems) according tostandard protocols. Genotyping success rates were 100%for the frozen tissues (44 tumors) and 98.5% for FFPE tissues(194/197), with 100% concordance among duplicates.

Expression analysis of indoleamine-2,3-dioxygenase inprostate tumors

Indoleamine-2,3-dioxygenase (IDO1) expression was mea-sured by quantitative RT-PCR using a Life Technologies Taq-Man expression assay (IDO1, Hs00984148_m1) and total RNAfrom prostate tumors of 21 AA and 22 EA men. Characteristicsof these patients have been described previously (12). In thiscohort, AA and EA patients are matched on Gleason score andpathologic stage. Relative normalized expression values werecalculated as described previously (25), using the group meanCt for the target and the endogenous control 18s RNA. Folddifferences were calculated as 2�DDCt. Graphs were preparedusing DCt ¼ Ct18S � Cttarget and plotted using GraphPad Prism 7.

Measurement of tryptophan in plasma samplesLevels of tryptophan, which is metabolized by IDO1,

were measured in plasma samples from randomly selected 50AA and 50 EA population-based controls (mean age: 63.4 and63.7, respectively) and 50 AA and 50 EA patient with prostatecancer (mean age: 61 and 62.4, respectively). These subjectswere previously recruited into the NCI-Maryland Prostate

Cancer Case–Control study (26). Tryptophan metabolite con-centrations were measured at SAIC/Leidos-NCI Frederick,Frederick, MD, using a described high-performance liquidchromatography method (27). Tryptophan measurementswere obtained for 197 of 200 plasma samples (98.5%).

In vitro induction of an IFN signature by overexpression ofIFNL4 in human prostate cancer cell lines

To examine IFNl4-induced gene expression, the humanprostate cancer cell lines, 22Rv1, PC-3, and MDA-PCa-2b, weretransfected with a previously described IFNL4 expression con-struct that generates IFNl4 protein with C-terminal Halo-tag(20). A GFP reporter gene was used to monitor transfectionefficiency. Transfection of the IFNL4-Halo construct and emptyHalo-control vector was performed in triplicates using Lipo-fectamine/LTX and yielded robust overexpression of IFNL4 inthe IFNL4-Halo–transfected cell lines without affecting cellviability (Supplementary Fig. S1). The IFNL4 qRT-PCR wasperformed as described previously (20). In the experimentswith 22Rv1 cells, we also added antibodies that block theactivity of IFNl4 - 20 mg/mL of goat anti–a-IL10R2 antibody(R&D Systems) that blocks one receptor of IFNl4, or 20 mg/mLof a rabbit monoclonal anti-IFNl4 antibody (Abcam,ab196984), that blocks IFNl4 directly. Cells were lysed after48 hours and total RNA was extracted with the RNeasy Kit withDNase I treatment (QIAGEN). One to 1.5 mg of total RNA wasused to generate cDNA using the RT2 First Strand Kit (QIA-GEN). The Human Type I IFN Response PCR array (QIAGEN)was used to evaluate expression of a panel of other relevantgenes. This array includes 96 SYBR Green expression assays fortype I IFNs (IFNA and IFNB) and their receptors, ISGs andmolecules involved in response and resistance to signaling, aswell as positive and negative controls. Differences in expressionbetween cells transfected with the IFNL4 construct and emptyvector were evaluated with a multiple comparisons-adjustedtwo-tailed t test. We also examined expression of two other typeIII IFNs to IFNL1 (Hs00601677_g1) and IFNL3 (Hs04193050),and endogenous control 18s RNA (4319413E) with Life Tech-nologies TaqMan expression assays. For all TaqMan assays,gene expression was measured in Ct values and normalized byendogenous control 18s RNA. Analysis of the PCR Arrays wasperformed using the GeneGlobe Data Analysis Software, inrelation to a panel of positive and negative controls includedon the array.

BrdU assayCells were preseeded in 96-well cell culture plates (Corning Life

Sciences) overnight and then cultured in either the completeculture medium or the medium without FBS, or transfected witheither the IFNL4-Halo expression vector or the vector controlconstruct. After 48-hour incubation, cells were assessed for cellproliferation using the bromodeoxyuridine (BrdU) colorimetricELISA assay (Roche) according to the provided protocol. Eightreplicates were analyzed per condition, and proliferation wascalculated as relative ratio by normalization BrdU incorporationto the control.

Statistical analysisAnalyses were conducted using SAS 9.2 (SAS Institute) and R

(R Foundation for Statistical Computing; http://www.r-project.org/). All statistical tests were two-sided. P < 0.05 was

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considered statistically significant. The nonparametric Mann–Whitney test was used for group comparisons with continuousdata and the Fisher exact test for categorized data. Associationsbetween a genotype and the occurrence of IRDS were estimatedwith linear regression models. Survival analysis was conductedwith a multivariate Cox proportional hazards regression modelunder a log-additive hazards assumption. Disease-free survivalwas defined from the date of prostatectomy to the date ofrecurrence (PSA defined). Overall survival was defined from thedate of the prostate cancer diagnosis to the date of death(Cleveland Clinic cohort). Survival analyses (disease-free, over-all) were controlled for age (<65 or �65 years), stage (I, II or III,IV) and Gleason score (<7 or �7). Statistical significance wasdetermined using the Wald test.

ResultsIRDS is prevalent in prostate tumors of AA men

We previously described an immune-inflammation signaturein tumors of AA patients that showed upregulation of genesinvolved in IFN signaling (12). Here, we examined whether twoIFN signatures, IRDS and IRG, are detectable in prostate tumorsand more prevalent in AA than EA patients. IRDS and IRG havebeen described independently but show considerable similarityand could be functionally equivalent, and are both associatedwith decreased breast cancer survival (16, 23). Our analysis foundthat both signatures are detected about twice as often in tumorsfrom AA than EA men in two independent datasets, Wallace andcolleagues' and Taylor and colleagues' (Fig. 1A and B). Forexample, IRDS was detected at a frequency of 67% and 42% inAA men in these two datasets, which is significantly higher thanthe observed frequency in EA men (33% and 18%, respectively).We also investigated IRDS and IRG in cultured prostate cancer

epithelial cells from 14 AA and 13 EA men. The expression datawere previously publishedby Timofeeva and colleagues (28). Thisanalysis showed that the two signatures can be detected in 5 of14 (36%) cell cultures fromAAmenand2of 13 (15%) for EAmen(Supplementary Fig. S2).

Of the two signatures, IRDS and IRG, only IRDS has beencharacterized (16, 17, 29). We therefore focused on IRDS in ourfollow-up analyses. Although IRDS captures the expression ofmany ISGs, others are not represented by this signature. BecauseIDO1 is an immunosuppressive ISG with a key function in cancerbiology, but not an IRDS gene (Supplementary Table S2), weexamined its expression in prostate tumors from 21 AA and 22EA patients from the Wallace and colleagues' dataset using aqRT-PCR approach. The expression analysis showed an approx-imately 2-fold increased tumor expression of IDO1 in AAmen when compared with EA men (Fig. 1C), consistent with the2-fold higher prevalence of IRDS in AA men. Because IDO1 usestryptophan as a substrate for its enzymatic activity, we examinedblood tryptophan levels in AA and EA patients with prostatecancer to assess whether increased tumor IDO1 in AA patientsmay affect these levels (Fig. 1D). We found very similar meanblood tryptophan levels in AA men (31.9 � 8.1 mmol/L) and EAmen (32.1 � 5.9 mmol/L) without prostate cancer, and in EApatients with prostate cancer (31.9 � 4.5 mmol/L), but a modestdecrease in blood tryptophan (by 8%–9%) among the AA cases(29.3� 7.6 mmol/L; P¼ 0.02 when compared with EA cases; P¼0.08 when compared with AA controls).

IRDS is associated with early disease recurrenceTo examine whether IRDS is clinically relevant for patients

with prostate cancer, we analyzed available gene expression andcancer recurrence data for 491 patients with prostate cancer in

Figure 1.

Increased frequency of two IFN signatures, IRDSand IRG, in prostate tumors from AA men.A, Analysis of 33 and 36 tumors from AA and EAmen, respectively, in the Wallace and colleagues'dataset (GSE6956). Prevalence of IRG and IRDS:55% and 67% in AA and 19% and 33% in EA.Permutated P value and FDR for difference in IRDSfrequency between AA and EA tumors: P ¼ 1.6 �10�4; FDR ¼ 3.7%. Red, upregulated expression.B, Analysis of 24 and 98 tumors from AA and EAmen, respectively, in the Taylor and colleagues'dataset (GSE21032). Prevalence of IRG and IRDS:46% and 42% in AA and 17% and 18% in EA.Permutated P value and FDR for difference in IRDSfrequency between AA and EA tumors: P ¼ 0.006;FDR ¼ 21%. Gene expression data were analyzedwith the SLEPR method, as described underMaterials and Methods. C, Increased mRNAexpression of IDO1 in prostate tumors from AAmen. IDO1 TaqMan assay was used to compareexpression in AA versus EA men. Lines ¼mean DCt

values. Fold expression difference is derived from2�DDCt values. D, Decreased abundance oftryptophan in plasma of AA patients with prostatecancer. P values from two-sided Mann–Whitneytest. Shown are means � SD.

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TCGA. We defined the occurrence of IRDS based on 45 IRDSgenes with measurable expression and used the RNA-seq datato categorize patients into 3 groups with low, medium, andhigh IRDS expression in their tumors (Fig. 2). The Kaplan–Meier survival plot shows that an increased IRDS expression isassociated with decreased disease-free survival (Fig. 2C). Usinga Cox regression model, we estimate that patients with highIRDS expression in their tumors have a 2-fold increased hazardof an earlier disease recurrence [HR ¼ 2.09; 95% confidenceinterval (CI), 1.07–4.10 after controlling for age, disease stage,and Gleason score], when compared with patients with lowIRDS expression.

IFNL4 rs368234815-DG is associated with IRDS and a distinctexpression profile in prostate tumors

IFNl4 is a recently discovered type III IFN that inducesa similar set of ISGs that constitute IRDS, and impairs anantiviral response, as shown for HCV (20). Production ofIFNl4 is genetically controlled by a genetic variant, IFNL4rs368234815-DG, which is the most common among subjectsof African ancestry (20). Thus, we examined whether IFNL4rs368234815-DG is associated with the occurrence of IRDS in44 prostate tumors that had available information on IRDSand the IFNL4 rs368234815-DG/TT genotype. As shown inTable 1, homozygote carriers of the DG allele (DG/DG) weresignificantly more likely to have IRDS-positive tumors thancarriers of the combined TT/TT and TT/DG genotypes. Whenwe restricted the analysis to the 23 AA patients in this cohort, weagain found a significant association between the DG/DG geno-type and the occurrence of IRDS in prostate tumors (OR ¼ 8.2;95% CI, 1.1–60.4). Of note, only AA men were carriers of theDG/DG genotype among the 44 men.

Further examination of the gene expression data revealedthat the IFNL4 rs368234815-DG is associated with increasedexpression of immune-, host defense-, and inflammation-related genes in prostate tumors (Fig. 3). Gene expressiondifferences based on the DG genotype separated tumors intothree distinct clusters. Cluster 1 was significantly enriched fortumors from AA men and carriers of the DG/DG genotype andshowed DG allele dosage increase of expression of genes suchas PTPRC, TNF, RSAD2, IFI44, NLRP3, CCL5, STAT1, CCL4,IFI35, IRF9, ISG15, IFNG, and MX1, among others. Many ofthese are IRDS genes (e.g., IFI35, IFI44, MX1, and STAT1). Thisobservation was further explored by a pathway analysis usingGO biological processes, molecular function, and cellularcomponent annotations. The approach identified GO termssuch as immune response, defense response, and response tovirus, as being associated with IFNL4 rs368234815-DG (FDR <5%). This observation was corroborated by transient over-expression of IFNL4 in 22Rv1, PC-3, and MDA-PCa-2b humanprostate cancer cells. Expression of IFNl4 induced an IFNsignature in these cells, consistent with IRDS, under theseexperimental conditions (Fig. 4A–C), although not affectingcell growth (Supplementary Fig. S1). The IFNL4-transfectedcells showed induction of known ISGs that are common toboth IRDS and IRG, such as IFIT1-3, IFIH1, IFITM1, OAS1,OAS2, MX1, IRF7, and IRF9, when compared with vectorcontrol–transfected cells. Furthermore, when we inhibitedIFNl4 signaling with blocking antibodies targeting either theIFNl4 receptor, IL10R2, or IFNl4, this signature was attenu-ated (Fig. 4A).

IFNL4 rs368234815-DG is associated with overall survival of AApatients with prostate cancer

To test whether IFNL4 rs368234815-DG may influenceeither disease-free or overall survival of patients with prostatecancer, we genotyped 194 AA prostatectomy patients fromthe Cleveland Clinic, as described in Materials and Methods.Twenty-five (12.8%) of the patients carried the TT/TT geno-type, 92 (47.4%) the TT/DG genotype, and 77 (39.7%)were homozygote for DG, which corresponds to a 63.4%DG allele frequency. These genotypes did not associate withthe Gleason score of tumors, nor did we find an associationwith disease recurrence (P ¼ 0.88) in this cohort. However,the DG/DG genotype was associated with a significantlydecreased overall survival of these patients (Fig. 4D). TheCox regression model showed that each copy of the DG alleleincreased the risk of death by about 2-fold (HR ¼ 2.07; 95%CI, 1.14–3.75 adjusted for age, disease stage, Gleason score;Ptrend ¼ 0.01) compared with carriers of the TT/TT genotype(Table 2).

DiscussionIn this study, we show that a subset of prostate tumors man-

ifests a distinct IFN signature, IRDS, that has previously beenlinked to acquired resistance to radiation and chemotherapy,increased metastases, and poor survival of patients with breastcancer and glioblastoma (16–19, 29, 30). We show that inpatients with prostate cancer, IRDS predicts decreased disease-free survival and is significantly more prevalent in tumors of AAthan EA patients. Furthermore, we discovered that a germlinevariant in the IFNL4 gene was significantly associated with theincreased prevalence of IRDS and all-cause mortality in AAprostatectomy patients, suggesting genetic predisposition to theseclinical outcomes.

Upregulated IFN signaling through the JAK–STAT signaling ispart of IRDS and the antiviral response (19), but is also triggeredby DNA methyltransferase inhibitors (31) and occurs in varioustypes of cancers (16, 30, 32, 33). In human triple-negative breastcancer, coinactivation of p53 and ARF coincides with an onco-genic IFNb–STAT1–ISG15 signaling signature that is reminiscentof IRDS (33). However, in primary prostate tumors, the inacti-vation of p53 and ARF is rather uncommon (34, 35), indicatingthat the common occurrence of IRDS in primary tumors of AApatients is likely caused by another mechanism. Investigationsusing mouse models demonstrated that IFN signaling throughSTAT1 is protumorigenic in leukemia and colon cancer develop-ment through chronic inflammation–mediated carcinogenesis(36, 37). These findings are consistent with our previous reportsthat tumors of AA patients demonstrate a prominent immune-inflammation signature that may increase tumor aggressivenessand can be targeted with anti-inflammatory drugs (12, 26). In astudy by Hardiman and colleagues (15), the effect of vitamin Dsupplementation on the prostate cancer transcriptome was inves-tigated in 10 AA and 17 EA patients. In line with our findings, theauthors found an immune-inflammation signature in tumors ofAA patients but also showed that this signature is diminished byvitamin D treatment.

Besides increasing inflammation and disease progression, theimmune-inflammation signature in AA prostate tumors maycause resistance to radiation and chemotherapy, and T-cellexhaustion. IRDS cooccurs as part of this immune-

Interferon Signature and African-American Prostate Cancer

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Figure 2.

IRDS is associated with early disease recurrence in the TCGA prostate cancer cohort. A, Expression of 45 IRDS genes identifies prostate tumors withlow (group 1: n ¼ 159), medium (group 2: n ¼ 270), and high (group 3: n ¼ 62) expression of IRDS (sum Z-score). Hierarchical clustering based on geneexpression of IRDS in 491 TCGA prostate tumors. Red, upregulated expression. B, Boxplots representing the relationship between IRDS expression andthe three groups. Grouping based on IRDS sum Z score statistics. C, High IRDS expression in prostate tumors is associated with decreased disease-freesurvival. Kaplan–Meier survival analysis. Log-rank test (P < 0.05) and unadjusted HR from a Cox regression analysis with Ptrend.

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Table 1. IFNL4 rs368234815-DG allele is associated with occurrence of IRDS in prostate tumors

IFNL4 genotype, N (%) Fisher's exact test OR

All tumors, N ¼ 44 TT/TT or TT/DG DG/DG P Adjusted OR (95% CI)a

IRDS-negative 23 (92%) 2 (8%) < 0.001 15.7 (2.7–90.6)IRDS-positive 8 (42%) 11 (58%)

Only tumors from AA men, n ¼ 23IRDS-negative 6 (75%) 2 (25%) 0.04 8.2 (1.1–60.4)IRDS-positive 4 (27%) 11 (73%)

aAdjusted for age at diagnosis and pathological stage.

Figure 3.

IFNL4 rs368234815-DG is associated with a distinct expression profile in prostate tumors (n ¼ 44). Hierarchical clustering based on expression of 1,194transcripts yields three distinct clusters and shows a significant association between IFNL4 rs368234815-DG and the pattern of gene expression (red,upregulated genes). IFNL4 rs368234815 genotypes are shown above the heatmap in red for DG/DG, green for TT/DG, and blue for TT/TT. Cluster 1 issignificantly enriched for tumors from AA men and carriers of the DG/DG genotype. Frame marks 511 probesets, representing 447 unique genes,with signal enrichment in cluster 1. GO relationships for these genes are shown to the right and include immune response, defense response andresponse to virus (FDR < 5% for all associations). The 1,194 transcripts were selected because their expression was associated with DG allele (P < 0.05)using a linear regression model where relationships with DG were analyzed under an additive model (coded as 0, 1, and 2 DG alleles).

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inflammation signature and has been shown to increase resis-tance to radiation (29), although inhibition of the JAK–STATpathway was found to result in resensitization of docetaxel-resistant DU145 prostate cancer cells (38). Others found that

the JAK–STAT signaling increases cross-resistance in myelomacell lines (39). More recently, several investigations linkedIRDS-like signatures to the clinical response to anti-CTLA4therapy and PD-1 blockade (40, 41). Accordingly, these

Figure 4.

IFNL4 overexpression induces an IFN signature in vitro and IFNL4 rs368234815-DG associates with overall survival. IFNl4 induces an IFN signature in the22Rv1 (A), PC-3 (B), and MDA-PCa-2b (C) human prostate cancer cells. Shown are heatmaps of genes related to the human type I IFN response aftertransfection of cells with an IFNL4 expression construct. Red, upregulated expression. In A, blocking antibodies were added to the culture mediumtargeting the IL10R2 receptor and IFNl4. �Significantly different gene expression induced by IFNL4 overexpression versus control. D, Association of IFNL4rs368234815-DG with overall survival among AA patients with prostate cancer (n ¼ 194). Carriers of the DG/DG genotype experienced the worstsurvival. Kaplan–Meier survival curve by IFNL4 genotype. Log-rank test: P < 0.05.

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signatures cause immunosuppression in melanomas and resis-tance to anti-CTLA4 therapy but increase the response rate toPD-1 blockade. PD-L1 is an ISG and an important mediator ofresistance to anti-CTLA4 therapy (40). PD-L1 expression maydetrimentally affect AA patients with prostate cancer because ofIRDS. Indeed, it was recently shown that expression of PD-L1 isincreased in prostate tumors of AA men, when compared withEA men, using IHC (42). We did not find that PD-L1 wassignificantly upregulated at the transcript level in AA prostatetumors in the Wallace and colleagues and TCGA datasets,suggesting that IRDS may regulate protein stability rather thanexpression of PD-L1 in these tumors. However, we found thatIDO1, another ISG, is upregulated on the mRNA level in AAprostate tumors, consistent with an immunosuppressive envi-ronment in these tumors. Additional measurements of plasmatryptophan levels in our study suggest that AA patients withprostate cancer may have a higher turnover of this IDO1substrate, although the reduction of plasma tryptophan wasrather modest.

Currently, there is no firm evidence that a viral infection is acause of prostate cancer (43), although a meta-analysis of 26tissue-based case–control studies reported an associationbetweenhuman papillomavirus positivity and the disease (44). In addi-tion, RNASEL, a gene that protects against viral pathogens, mayhave an important tumor suppressor function in prostate cancer(45, 46). Besides viral infections, spontaneous reactivation ofendogenous retroviruses may also lead to occurrence of IRDS. Wereported the upregulation of the human endogenous retrovirustype K envelope protein in prostate tumors of AA patients (47).Moreover, it was recently shown that treatmentwithDNAmethyl-transferase inhibitors can lead to reactivation of endogenousretroviruses and an IRDS-like signature in patients with melano-ma that increases the sensitivity to immune checkpoint therapy(31). Western diet may also upregulate IFN signaling (48).

With this study, we provide the first evidence that IFNL4rs368234815-DG could be a predisposition factor for IRDS inpatients with prostate cancer. Although this effect is not exclu-sive to AA men, but based on a much higher DG allelefrequency in AA compared with EA patients (e.g., 62% in AAvs. 34% in EA in the NCI-Maryland Prostate Cancer Case–Control Study), this genetic predisposition would be mostimportant for men of African ancestry. This stark differencein the DG allele frequency between AA and EA patients couldaccount for some of the known health disparity in outcomesof prostate cancer. IFNl4 protein is only produced in indivi-duals with IFNL4 rs368234815-DG and attenuates antiviralresponses through negative regulation of IFN signaling (21).

We showed that IFNl4 induces an IFN signature in threehuman prostate cancer cell lines, but could not detect IFNL4expression in human prostate tumors based on TCGA RNA-seqdata. IFNL4 expression is known to be low and transient, butIFNl4 is potent even at very low levels (21, 49). Finally, IFNL4rs368234815-DG was associated with overall survival of AAprostatectomy patients, indicating a broader biological effectof this variant. We also found similar association with overallsurvival of AA prostatectomy patients for an intronic IFNL4variant, rs12979860-T, which is in high linkage disequilibriumwith rs368234815-DG (Supplementary Fig. S3). However, incontrast to IRDS, we did not find an association of IFNL4rs368234815-DG with decreased disease-free survival. Perhaps,our analysis of 194 genotyped AA prostatectomy patients wasunderpowered to detect this association, or the impact ofIFNL4 rs368234815-DG on disease recurrence is weaker thanthe effect of IRDS. Alternatively, IFNL4 rs368234815-DG maymainly affect outcomes of late-stage therapies such as chemo-therapy and immunotherapy, or may have a detrimentalimpact on morbidities arising from long-term androgen abla-tion therapy, explaining the observed association of this geno-type with overall survival.

Thus, future research is needed to define the role of IFNl4 inIRDS among patients with prostate cancer. Yet, based on ourfindings, IFNL4 rs368234815-DGmay have potential clinical usefor identifying patients with prostate cancer with increased sen-sitivity to immune checkpoint blockade therapy and therebypredicting disease outcomes.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: W. Tang, C.A. Loffredo, R.H. Silverman, G.R. Stark,S. AmbsDevelopment of methodology: W. Tang, T.A. Wallace, C. Magi-Galluzzi,S.V. Jordan, L. Prokunina-OlssonAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): W. Tang, C. Magi-Galluzzi, T.H. Dorsey,O.O. Onabajo, A. Obajemu, C.A. Loffredo, E.A. KleinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Tang, M. Yi, C.A. Loffredo, R.M. Stephens,L. Prokunina-Olsson, S. AmbsWriting, review, and/or revision of the manuscript: W. Tang, T.A. Wallace,M. Yi, C. Magi-Galluzzi, S.V. Jordan, C.A. Loffredo, R.H. Silverman, G.R. Stark,E.A. Klein, L. Prokunina-Olsson, S. AmbsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W. Tang, T.H. DorseyStudy supervision: S. Ambs

Table 2. IFNL4 rs368234815-DG allele is associated with decreased overall survival of AA patients with prostate cancer

Risk allele RAF, % Genotype N (%) Crude HR (95% CI) Adjusted HR (95% CI)a

rs368234815-DG 63.4 TT/TT 25 (12.9) 1.00 (reference) 1.00 (reference)TT/DG 92 (47.4) 1.47 (0.32–6.75) 2.07(0.44–9.66)DG/DG 77 (39.7) 3.26 (0.75–14.15) 4.28 (0.97–18.90)Total 194

per allele — 2.00 (1.09–3.67) 2.07 (1.14–3.75)Ptrend — 0.02 0.01

DG/DG vs. TT/TTþTT/DG — 2.39 (1.14–5.01) 2.21 (1.05–4.69)P — 0.02 0.02

NOTE: RAF ¼ risk allele frequency in dataset (n ¼ 194).aMultivariable Cox regression model adjusted for age, disease stage (TNM), and Gleason score; Ptrend: DG/DG versus TT/DG versus TT/TT.

Interferon Signature and African-American Prostate Cancer

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AcknowledgmentsThis research was supported by the Intramural Research Program of the

Center for Cancer Research (ZIA BC010499 and ZIA BC010624, to S. Ambs)and Division of Cancer Epidemiology and Genetics (ZIA CP010201, toL. Prokunina-Olsson), NCI, NIH, the Prostate Cancer Foundation 2013 BenFranklin-PCF Special Challenge Award (G.R. Stark, S. Ambs, and E.A. Klein),the Cleveland Clinic Prostate Cancer Center of Excellence Award (toR.H. Silverman, G.R. Stark, C. Magi-Galluzzi, and E.A. Klein), and theNational Institute of Allergy and Infectious Diseases (NIAID), NIH grantR01AI135922 (to R.H. Silverman). We thank the Cooperative ProstateCancer Tissue Resource (CPCTR) for providing tissue specimens and sup-

porting data. We would also like to thank personnel at the University ofMaryland and the Baltimore Veterans Administration Hospital for theircontributions with the recruitment of subjects.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 5, 2018; revised June 12, 2018; accepted July 10, 2018;published first July 16, 2018.

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CancerTumors and Survival of African-American Men with Prostate G Allele Is Associated with an Interferon Signature in∆-IFNL4

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