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A Multigene Assay Determines Risk of Recurrencein Patients with Triple-Negative Breast CancerRachel L. Stewart1, Katherine L. Updike2, Rachel E. Factor3, N. Lynn Henry4,Kenneth M. Boucher5, Philip S. Bernard3, and Katherine E. Varley2

Abstract

Approximately 40% of patients with stage I–III triple-neg-ative breast cancer (TNBC) recur after standard treatment,whereas the remaining 60% experience long-term disease-freesurvival (DFS). There are currently no clinical tests to assess therisk of recurrence in TNBC patients. We previously determinedthat TNBC patients with MHC class II (MHCII) pathwayexpression in their tumors experienced significantly longerDFS. To translate this discovery into a clinical test, we devel-oped an MHCII Immune Activation assay, which measuresexpression of 36 genes using NanoString technology. Preana-lytical testing confirmed that the assay is accurate and repro-ducible in formalin-fixed paraffin-embedded (FFPE) tumorspecimens. The assay measurements were concordant withRNA-seq, MHCII protein expression, and tumor-infiltratinglymphocyte counts. In a training set of 44 primary TNBCtumors, theMHCII Immune Activation Score was significantlyassociated with longer DFS (HR ¼ 0.17; P ¼ 0.015). In an

independent validation cohort of 56 primary FFPE TNBCtumors, the Immune Activation Score was significantly asso-ciatedwith longerDFS (HR¼ 0.19; P¼ 0.011) independent ofclinical stage. An Immune Activation Score threshold foridentifying patients with very low risk of relapse in the trainingset provided 100% specificity in the validation cohort. Theassay format enables adoption as a standardized clinicalprognostic test for identifying TNBC patients with a low riskof recurrence. Correlative data support future studies to deter-mine if the assay can identify patients in whom chemotherapycan be safely deescalated and patients likely to respond toimmunotherapy.

Significance: The MHCII Immune Activation assay identi-fies TNBC patients with a low risk of recurrence, addressing acritical need for prognostic biomarker tests that enable preci-sion medicine for TNBC patients.

IntroductionTriple-negative breast cancer (TNBC) is a clinical subtype of

invasive breast cancer that is defined by the absence of standardmarkers used for prognosis and treatment decisions [estrogenreceptor (ER), progesterone receptor (PR), and HER2]. TNBC isnotable for its aggressive behavior and high rates of local anddistant recurrence (1). TNBC patients are treated with localtherapy and cytotoxic chemotherapy. Patient outcomes aredisparate. Approximately 42% of patients experience rapidrelapses with a peak at 3 years from diagnosis, whereas theremaining 58% of patients have long-term disease-free survival

(DFS; ref. 2). Physicians cannot currently predict which patientswill relapse, even after intensive chemotherapy, and whichpatients will have long-term DFS and might do equally wellwith deescalation of their chemotherapy regimen. Currently,most TNBC patients are treated with aggressive chemotherapy,which can result in serious long-term toxicity including per-manent peripheral neuropathy, cardiac toxicity, and secondarymalignancies (3–8). A current goal of the TNBC biomarker fieldis to develop clinical tools that can be used to identify patientswho do not require aggressive treatment and can be spared theassociated toxicities.

We recently reported that expression of the MHC Class IIantigen presentation pathway (MHCII) in TNBC tumor cells issignificantly associated with long-term DFS (9). Further, highMHCII expression in tumor cells was associated with thepresence of tumor-infiltrating lymphocytes (TIL; ref. 9), whichare known to be associated with good prognosis in patientswith TNBC (10–14). An independent research team performedIHC on 681 TNBC patient tumors and confirmed that highexpression of MHCII in tumor cells was associated with largeamounts of tumor-infiltrating CD4- and CD8-positive T cells,and longer DFS (15). Mouse studies have shown that MHCIIexpression on tumor cells triggers T-cell recruitment and inhi-bits tumor progression (16–23). A standardized method for themorphologic evaluation of TILs in patient tumor samples hasbeen developed, but has not entered routine clinical prac-tice (24, 25). Although promising, broad clinical implementa-tion of this method may be limited by pathologist training,interobserver variability, and time required for assessment (26).

1Department of Pathology and Laboratory Medicine and the Markey CancerCenter, University of Kentucky College of Medicine, Lexington, Kentucky.2Department of Oncological Sciences, Huntsman Cancer Institute, Universityof Utah, Salt Lake City, Utah. 3Department of Pathology, University of Utah/Huntsman Cancer Institute, Salt Lake City, Utah. 4Department of InternalMedicine, University of Utah School of Medicine, Salt Lake City, Utah. 5StudyDesign andBiostatistics Center, School ofMedicine, University of Utah, Salt LakeCity, Utah.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Katherine E. Varley, University of Utah HuntsmanCancer Institute, 2000 Circle of Hope, Room 3719, Salt Lake City, UT 84112.Phone: 801-213-5661; Fax: 801-585-6410; E-mail: [email protected]

Cancer Res 2019;79:3466–78

doi: 10.1158/0008-5472.CAN-18-3014

�2019 American Association for Cancer Research.

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Furthermore, this approach does not discern lymphocyte sub-sets or T-cell activation states (24, 25).

Although histologic assays for several MHCII proteins and TILcounting could be combined to develop diagnostic criteria, theprocess would be complex. Historically, multiplexed IHC assays(e.g., IHC4) have not performed as well as multiplexed geneexpression assays (27, 28). Compared with traditional pathologicscoring systems, a multiplexed gene expression test can measurethe expression ofmany genes in theMHCII pathway, quantify TILmarkers simultaneously, and has a larger dynamic range ofmeasurements with finer resolution.

In routine clinical practice, patients' tumors are collected andprocessed as formalin-fixed, paraffin-embedded (FFPE) tissues,which results in significant degradation of mRNA (29). PCR wasthe first technology used to demonstrate that small fragmentedRNA transcripts could be recovered from FFPE tissue and used toaccurately quantify gene expression in breast tumors (30). Thisenabled the development of the first gene expression prognosticassay for patients with hormone receptor–positive (HRþ) breastcancer (Oncotype Dx; ref. 31). There are now several gene expres-sion assays that are indicated for use in patients with HRþ breastcancer (32–37); however, there are no clinically validated assaysavailable for patients with TNBC.

TheNanoString nCounter platform is an alternativemethod formeasuring gene expression in clinical FFPE specimens. Nano-String nCounter technology is unique in that it measures RNAdirectly without amplification or cloning, which eliminates thebiases that can be introduced by other PCR or sequencing-basedmethodologies (38, 39). One clinical prognostic test for HRþ

breast cancer (Prosigna) utilizes NanoString technology (32, 37,40). NanoString obtained a CE Mark for its Prosigna assay in2012, followed by FDA clearance in September 2013. Prosigna isnow included in clinical oncology guidelines for themanagementof HRþ breast cancer (41) and is performed in qualified clinicallaboratories around the world. In this study, we leveraged thisprevious success in clinical assay development on the NanoStringnCounter platform to develop an assay for MHCII and TIL geneexpression that could be used to assess prognosis in TNBCpatients.

Materials and MethodsNanoString probe design

A custom panel of probes formeasuring expression of 36 geneson the NanoString nCounter platform was designed. Probesequences were compared with RNA-seq data from TNBCtumors (9) to confirm that mRNA isoforms in TNBC would bedetected by the probe sequences, and redesigned as necessary.The probe sequences were then synthesized by Integrated DNATechnologies, Inc. The probe A oligos were purified using highperformance liquid chromatography, and the Probe B oligoswere purified using polyacrylamide gel electrophoresis. The fullsequence of the probes is provided in Supplementary Table S1.

NanoString nCounter assayWe used NanoString nCounter Elements TagSets and Master

Kits to develop the assay. Custom gene-specific oligonucleotideprobes (Probe Sequence in Supplementary Table S1) were pro-ducedby IntegratedDNATechnologies.Hybridization and count-ing were performed according to the manufacturer's specifica-tions. Briefly, gene-specific probes were hybridized with Nano-

String Elements TagSets and RNA at 67�C for 24 hours. Afterhybridization, samples were transferred to the automated nCoun-ter Prep Station for purification and immobilization onto thesample cartridge. After sample preparation was complete, thesample cartridge was transferred to the nCounter Digital Analyzerfor imaging and analysis. All samples were analyzed using themaximum resolution setting (555 images per sample).

Approval for use of patient specimensApproval for the useof archival tissue specimenswas granted by

Institutional Review Boards (IRB) at the University of Utah andthe University of Kentucky. The research was conducted in accor-dance with recognized ethical guidelines including the U.S. Com-mon Rule. Written-informed consent was obtained for fresh-frozen tissue collections. For previously collected archival FFPEblocks, the IRBs waived the requirement for informed consent.

RNA from frozen tissuesRNA remaining from frozen tissue collected for previous stud-

ies was used (9, 42). The RNA-seq data from these samples arepublicly available through GEO Accession GSE58135. For thecomparison of frozen and FFPE sections from the same tumor,frozen breast cancer specimenswere obtained from theUniversityofKentuckyMarkeyCancerCenter BiospecimenProcurement andTranslational Pathology Shared Resource Facility (BPTP SRF).These tissues were collected from breast surgical specimens underIRB protocols # 04-0454 and 11-0750. Fresh-frozen breast tissueswere embedded in Tissue-TekO.C.T. Compound (Sakura Finetek)and sectioned at �20�C on a cryostat. An initial 4 mm tissuesection was cut and stained using hematoxylin and eosin (H&E)so that tumor cellularity could be assessed by a pathologist. Onlycases with �10% tumor cellularity were included. After assessingthe H&E slide, a pathologist cut an additional 10 unstainedsections at 10 mm each. Unstained sections were collected in lysisbuffer and homogenized in a bullet blender (NextAdvance); RNAwas then isolated using an E.Z.N.A RNA Isolation Kit (OmegaBio-tek). After frozen sections had been taken for RNA isolation,the remnant block was taken off the cryostat, placed in a tissuecassette, and submitted for routine processing and embedding(creation of an FFPE block) in a pathology laboratory.

FFPE sample identificationThis project was performed under an approved University of

Utah IRBprotocol (#24487).Natural language searcheswere usedto identify surgical pathology cases with a diagnosis of invasivecarcinoma of the breast. Only breast tumors from patients withprimary stage I–III breast cancer were included in the study.Surgical pathology reports were reviewed by a pathologist todetermine ER, PR, and HER2 status. Only TNBC cases withpretreatment tumormaterial available in the archiveswere includ-ed. Detailed clinicopathologic, stage, and outcome data wereobtained through review of the pathology report and medicalrecord. DFS was defined as the length of time that the patientsurvived after a primary diagnosis of breast cancer without anyevidence of local disease recurrence or distant metastases. Eventsincluded ipsilateral breast recurrence and distant metastases.

Slide review, macrodissection, and RNA isolation from FFPEtissue

A pathologist reviewed all cases and selected the best FFPEblock from each case for analysis, taking care to avoid blocks with

A Multigene Assay to Assess Risk of Recurrence in TNBC

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low tumor cellularity, or with large areas of necrosis, calcification,or fibrosis. For each block, a fresh H&E-stained slide and adjacentunstained sections (10 mm) were obtained. A board-certifiedpathologist reviewed each H&E section and confirmed the pres-ence of invasive breast cancer. Tumorswere required to be�4mmin size and to have at least 10% tumor cellularity. Using theserequirements, only a single case was initially deemed inadequatedue to low tumor cellularity (

utilizing DAB (3-30 diaminobenzidine) as the chromogen.Tissue sections were counterstained with hematoxylin for 8minutes. The slides were removed from the immunostainer andplaced in a dH2O/DAWN mixture. The sections were gentlywashed in a mixture of deionized water and DAWN solution toremove any coverslip oil applied by the automated instrument.The slides were gently rinsed in deionized water until all of thewash mixture was removed. The slides were dehydrated ingraded ethanol, cleared in xylene, and then coverslipped. Forall staining runs, positive and negative controls were includedand stained appropriately in all cases. Benign human tonsil wasused as a positive control, whereas skeletal muscle was used as anegative control. In addition, positive staining in macrophagesand infiltrating lymphocytes served as internal positive controlsfor all cases. Scoring for HLA-DR and HLA-DR/DP/DQ wasperformed by a board-certified pathologist who was blinded toclinical variables. Expression of HLA-DR and HLA-DR/DP/DQwas assessed in tumor epithelial cells using a standard semi-quantitative system: negative (0), weak (1), moderate (2), andstrong (3).

ResultsA diagrammatic outline of this study's design and analyses is

provided in Supplementary Fig. S2.

Design of the MHCII immune activation assayThemajor goal of this study was to develop amultiplexed gene

expression assay on theNanoString nCounter platform that couldaccuratelymeasure the expression ofMHCII andTIL genes in FFPETNBC tumor specimens. We have named this the "MHCIIImmune Activation" assay.

The MHCII Immune Activation assay uses custom gene-specific oligo probes designed to 36 genes including MHCIIsignature genes, TIL genes, Subtype Verification genes, andHousekeeping Control genes (Fig. 1A; Probe Sequences inSupplementary Table S1). The MHCII genes were selectedbased on significant association with longer DFS in the previ-ous study (9). CIITA is the master transcriptional transactivatorof the MHCII pathway and is required to induce expression ofthe other genes in the pathway (48, 49). Candidate TIL geneswere selected based on high Spearman correlation (R > 0.5)with CIITA expression in the TNBC tumors in the previousstudy (9) and membership in the Gene Otology classification"Positive regulation of T cell activation" (50–52). Nine can-didate genes that were identified as TIL markers in recentpublications were selected for the assay (53–55). The selectedTIL genes include markers of T-cell types, as well as markers ofT-cell activation, T-cell memory, and T-cell interactions withtumor cells. The Subtype Verification genes were previouslydetermined to be the best distinguishers of basal-like TNBCfrom other subtypes using the PAM50 gene set (56). During theanalytical/technical development of the PAM50 signature,statistical algorithms to identify the best housekeeping controlgene sets for normalization in breast cancer were developed byour group (57). The five best housekeeping control genes fornormalizing classifier genes across all types of breast cancer andacross different ages of FFPE procurement were selected for thisassay (57).

Preanalytical testing of the MHCII immune activation assayWe chose to develop the assay on the NanoString nCounter

platform because previous studies reported that the platformprovides accurate gene expression measurements even in degrad-ed RNA from FFPE specimens (39). To ensure that the MHCIIImmune Activation assay accurately measures gene expression inFFPE specimens, the MHCII Immune Activation assay was per-formed on three pairs of matched frozen and FFPE breast tumorspecimens.Measurementswere highly correlated (SpearmanR2¼0.89–0.96;P

Figure 1.

Preanalytical testing of the MHCII Immune Activation assay.A, Gene sets measured by the assay. B, The assay provided similar measurements of gene expressionin frozen and FFPE sections from the same tumor (n¼ 3). Each point in the scatter plot represents the expression values for one of 36 genes. C, The assayprovided highly similar gene expression measurements between two replicates of each of 11 different FFPE breast tumor RNA samples. Each point in the scatterplot represents the expression values for one of 36 genes in one of 11 samples. Each of the 11 samples is depicted in a different color. D, The TIL genes in the assaywere differentially expressed between histologically confirmed TIL-high and TIL-low TNBC tumors. E, The Subtype Verification genes in the MHCII ImmuneActivation assay were differentially expressed between FFPE tumor specimens previously classified by the PAM50 assay as basal-like (n¼ 8), luminal A (n¼ 8),luminal B (n¼ 8), and HER2-enriched (n¼ 9). F,A threshold chosen for the basal-like score distinguishes basal-like tumors from other subtypes.

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tissue samples (n ¼ 44) that had been previously analyzed usingRNA-seq (9). From each sample, 50 to 250 ng of RNA washybridized with the custom gene-specific probes and ElementsTagSets and analyzed on the NanoString nCounter AnalysisSystem. The gene expression counts in each sample were back-ground subtracted and normalized to housekeeping genes, asdescribed in Materials and Methods. Five samples were excludedfrom analysis because they did not meet the basal-like scorethreshold defined in the preanalytical testing. The remaining39 samples were analyzed for MHCII and TIL gene expression.

Three gene probes (HLA-DQA1, HLA-DRB5, and HLA-DRB6)were excluded from further analysis due to poor concordancebetween the RNA-seq and NanoString data (SupplementaryFig. S4). The remaining MHCII gene expression measurementsobtained from the MHCII Immune Activation assay and fromRNA-seq on the same samples were highly correlated (meanSpearman R2 ¼ 0.88, mean P ¼ 0.008, Fig. 2A). This resultconfirms the accuracy of this new MHCII Immune Activationassay on the NanoString nCounter instrument.

To determine if the MHCII Immune Activation assay coulddetect differential expression of MHCII genes between TNBCpatients who relapsed and those who did not, an "MHCII Score"for each sample was calculated, defined as the geometric mean ofthe MHCII gene expression values. MHCII scores were signifi-cantly higher (one-sided Mann–Whitney P ¼ 0.0022) in TNBCpatients who did not relapse compared with those who didrelapse (Fig. 2B). A Kaplan–Meier curve using a threshold forMHCII score that provides the most significant log-rank P valuedemonstrated that the MHCII Immune Activation assay repro-duced the significant prognostic difference between tumors withhigh and low MHCII expression (log-rank P ¼ 0.0045, Fig. 2C,threshold depicted in Fig. 2B). This result confirms that theMHCIIgene expression signaturemaintains its prognostic significance onthe Nanostring nCounter platform.

A heatmap of theMHCII and TIL genes in TNBCpatient tumorsdemonstrated that expression of MHCII and TIL genes is highlycorrelated within a tumor (Fig. 2D). Similarly, MHCII and TILscores were correlated across samples (Spearman R2 ¼ 0.71;Supplementary Fig. S5). To determine whether expression of theMHCII and TIL genes could be combined into score that could beused to assess prognosis, an Immune Activation Score for eachsample was calculated using the geometric mean of the MHCIIand TIL gene expression values. Immune Activation Scores weresignificantly higher (one-sided Mann–Whitney P ¼ 0.0041) inTNBC patients who did not relapse compared with those who didrelapse (Fig. 2E). A Kaplan–Meier curve using a threshold for theImmune Activation Score that provides the same Specificity(90%) as the MHCII score demonstrated that patients with highImmune Activation Scores have a significantly higher probabilityof DFS than those with low Immune Activation Scores (log-rankP ¼ 0.022, Fig. 2F, threshold ¼ 1,750 depicted in Fig. 2E). Thisresult confirms the prognostic power of the Immune ActivationScore generated by the MHCII Immune Activation assay.

Validation of the MHCII immune activation assay in anindependent cohort

The second major goal of this study was to validate that theMHCII Immune Activation assay could be used to assess prog-nosis in an independent institutional cohort of TNBC patients.Chart review was used to select cases that generally represent thediverse presentation and outcomes that are seen in TNBC patients

in clinical practice at the University of Utah (n ¼ 56). Selectedcases included age 35–70 (median, 55), stage I–III disease (major-ity stage II), tumor size T1–T4 (majority T2), histologic grade 2–3(majority grade 3), and patients with positive and negative lymphnodes (Supplementary Table S2). Overall, these demographicsand the number of cases are similar to the cohort used in theprevious study and the training set (Supplementary Table S2;ref. 9).

A board-certified anatomic pathologist selected clinical FFPEtissue blocks in which there was adequate tumor tissue formacrodissection. All specimens were collected prior to chemo-therapy. The MHCII Immune Activation assay was performed onRNA isolated from the TNBC FFPE specimens using a protocolsimilar to the Prosigna test, as described in detail in Materials andMethods.

Eleven samples were excluded from analysis because they didnot meet the basal-like score threshold defined in the preanaly-tical testing. The observation that not all TNBC tumors will beclassified into the basal-like subtype based on gene expression isconsistent with prior studies that report the presence of luminalandrogen receptor subtype tumors and HER2-enriched subtypetumors among TNBCs (59, 60). The remaining 45 samples wereanalyzed for MHCII and TIL gene expression.

The expression of MHCII and TIL genes was correlated withineach tumor, similar to the training set (Fig. 3A). MHCII and TILscores were also correlated across samples (Spearman R2 ¼ 0.58,Supplementary Fig. S5). The geometric mean of the MHCII andTIL gene expression values was used to calculate an ImmuneActivation Score for each sample. Immune Activation Scores weresignificantly higher (one-sided Mann–Whitney, P ¼ 0.0278) inTNBC patients who did not relapse compared with those who didrelapse (Fig. 3B). A Kaplan–Meier curve using the same ImmuneActivation Score threshold as the training set demonstrated asignificant prognostic difference between tumors with high andlow Immune Activation Scores (log-rank P ¼ 0.021, Fig. 3C,threshold ¼ 1,750 depicted as a dashed line in Fig. 3B). Thisresult confirms the prognostic significance of the MHCII ImmuneActivation assay in this independent cohort.

Assessing risk of recurrence using the MHCII immuneactivation assay

The most likely clinical use of the MHCII Immune Activationassay would be to identify patients that have a very low risk ofrelapse, and distinguish them from patients who have an averagerisk of relapse. To determine if the MHCII Immune ActivationAssay could be used to identify patients that have a very low risk ofrelapse, an ROC curve was calculated for the Immune ActivationScores in the training set and validation cohort (Fig. 4A, ROCstatistics are provided in Supplementary Fig. S6). This clinicalapplication of the assay requires high specificity to correctlyidentify patients who have a low risk of recurrence and avoidmisclassifying patients that may recur. To evaluate the specificityof the assay, threshold analysis of the ROC curve was used tocalculate the Immune Activation Score that results in 95% spec-ificity for identifying patients who do not relapse in the trainingset (threshold ¼ 2,400). The 95% confidence intervals (CI) forthe threshold that provides 95% specificity are depicted in theROC curve in Fig. 4A. When this Immune Activation Scorethreshold was applied to the validation cohort, the specificity foridentifying patients who did not relapse was 100%, i.e., 0 patientswith Immune Activation Scores above the threshold relapsed






Figure 2.

MHCII Immune Activation assay in a training set of TNBC tumors. A,MHCII gene expression measurements from the MHCII Immune Activation assay and RNA-seqon the same TNBC tumor samples were highly correlated. Each of the 10 genes is a different color. B,MHCII Scores were significantly higher in patients who didnot relapse. Mean and 95% CI are shown. Threshold is a dashed line, red circle classified as high, and blue circle classified as low. C,A Kaplan–Meier curve and log-rank P value show significantly longer DFS in patients with high MHCII Scores. D, Expressions of MHCII and TIL genes are highly correlated within TNBC patienttumors in the training set. E, Immune Activation Scores calculated using MHCII and TIL genes were significantly higher in patients who did not relapse. Mean and95% CI are shown. Threshold is dashed line, red circles classified as high, and blue circles classified as low. F, A Kaplan–Meier curve and log-rank P value showsignificantly longer DFS in patients with high Immune Activation Scores using the threshold depicted in E.

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(Fig. 4B). Kaplan–Meier curves were created using this ImmuneActivation Score threshold to stratify patients, which demon-strates the difference in probability of DFS in both the trainingset (Fig. 4C) and the validation cohort (Fig. 4D).

In multigene clinical tests used to assess prognosis in HRþ

breast cancer (e.g., Prosigna and Oncotype Dx), the results arecontinuous variables that are linearly related to a patient's risk ofrecurrence (27, 61). Currently, the quantitative results of thesetests are used to classify patients into groups of low, intermediate,and high risk of recurrence for clinical management. The ImmuneActivation Score produced by this assay is also a continuousvariable. To determine if the Immune Activation Score producedby this assay is linearly related to a patient's risk of recurrence, thecumulative risk of recurrencewas calculated for patients across therange of Immune Activation Scores observed in the training setand validation cohort. The risk of recurrence in both the trainingset and validation cohort is a linear function of the log10 ImmuneActivation Score (Fig. 4E). This result confirms that a patient's riskof recurrence is monotonically related to the Immune ActivationScore. In the future, larger studies could be used to define thresh-olds to classify TNBC patients into groups with low, intermediate,or high risk of recurrence.

Cox proportional hazards regression models were generated totest the association between DFS, clinical variables, and ImmuneActivation Score in the training set and validation cohort. Inunivariate Cox regression, Immune Activation Score and stage atdiagnosis were significantly associated with DFS in both the

training set and validation cohort (Table 1). The Immune Acti-vation Score hazard ratio (HR) was 0.1430 (95% CI ¼ 0.03683–0.5555) in the training set and 0.2111 (95% CI ¼ 0.06075–0.7335) in the validation cohort, indicating a good prognosticfactor. The HR for stage was 2.1227 (95% CI ¼ 1.439–3.131) inthe training set and 1.628 (95% CI ¼ 1.204–2.201) in thevalidation cohort, indicating a poor prognostic factor. The otherclinical parameters were not significantly associated with DFS,including age at diagnosis, and whether the patient receivedchemotherapy (Table 1). In the multivariable Cox proportionalhazards regression model for both the training set and thevalidation cohort, Immune Activation Score and stage at diagno-sis both remained significant, and their HRs were similar to thosein the univariate analysis (Table 1). This result indicates that theImmune Activation Score is an independent predictor of DFS,even when accounting for the differences in DFS associated with apatient's disease stage at diagnosis.

ACox proportional hazardsmodel of the effect of stage alone inthe validation cohort predicts that a patient diagnosed with stageIIB disease has a 59% probability of 5-year DFS. A Cox propor-tional hazards model including both stage and Immune Activa-tion score predicts that a stage IIB patient with a high ImmuneActivation Score of 4,000 has a 79% probability of 5-year DFS,whereas a patient with the same disease stage and a low ImmuneActivation Score of 400 has a 32% probability of 5-year DFS. Thissuggests that a clinical decision-making tool that incorporated theImmune Activation Score in addition to the patient's disease stage

Figure 3.

MHCII Immune Activation Scores inindependent validation cohort ofFFPE TNBC tumors. A, Expressionsof MHCII and TIL genes are highlycorrelated within TNBC patienttumors in the independentvalidation cohort. B, ImmuneActivation Scores calculated usingMHCII and TIL genes in the MHCIIImmune Activation assay weresignificantly higher in patients whodid not relapse. Mean and 95% CIare shown. Threshold is a dashedline, red circle classified as high, andblue circle classified as low.C,A Kaplan–Meier curve andlog-rank P value show significantlylonger DFS in patients with highImmune Activation Scores using thethreshold depicted in B.






Figure 4.

Using Immune Activation Scores to identify patients with a low risk of recurrence. A, ROC curve analysis of the training set was used to select an ImmuneActivation Score threshold that results in 95% specificity for identifying patients who do not relapse. Green, training set ROC curve. Orange, validation cohortROC curve. 95% CIs for the threshold that provides 95% specificity in training set shown as black error bars. B,When this Immune Activation Score threshold wasapplied to the independent validation cohort, the specificity for identifying patients who did not relapse was 100%. C, Kaplan–Meier curve that stratifies patientsin the training set based on the Immune Activation Score threshold that provides 95% specificity.D, Kaplan–Meier curve of the same threshold applied to theindependent validation cohort demonstrates longer DFS in patients with Immune Activation Scores above the threshold. E, Risk of recurrence can be modeled asa linear function of the log10 Immune Activation Score in both the training set and validation cohort.

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could provide improved assessment of a patient's risk of recur-rence. Further studies in larger cohorts will be needed to train andevaluate a predictive model that incorporates Immune Activationscore.

Comparison of MHCII immune activation assay with IHC andhistologic TIL counting

The results from the MHCII Immune Activation assay confirmthat elevated expressionofMHCII andTIL genes is associatedwitha significantly reduced risk of recurrence in TNBC patients. Todetermine if these gene expression measurements correlate withtraditional histologic assessment of MHCII expression and TILcounting, IHC andH&E staining was performed on FFPE sectionsfrom the specimens analyzed in the validation cohort, which wasreviewed by a board-certified anatomic pathologist who specia-lizes in breast pathology.

In tumors with the highest Immune Activation Scores,MHCII protein was strongly expressed in a membranous pat-tern within infiltrating carcinoma cells and in associated TILs(Fig. 5A). Tumors with an intermediate Immune ActivationScore showed variable MHCII expression; in these cases, stain-ing was often heterogeneous and of moderate intensity(Fig. 5A). In tumors with the lowest Immune Activation Scores,MHCII protein expression was absent in invasive carcinomacells and present only in rare tumor-associated inflammatorycells (Fig. 5A).

TIL quantification was performed using a histologic "goldstandard" protocol developed by a consensus committee on TILsin breast cancer (24, 25). The TIL Score measured by the MHCIIImmuneActivation assaywas highly correlatedwithmorphologicassessment of stromal TIL percentage (Spearman R2 ¼ 0.69, P <0.0001, Fig. 5B). These results confirm that the MHCII ImmuneActivation assay on the Nanostring nCounter provides a stan-dardized and multiplexed procedure for measuring MHCIIexpression and TILs in FFPE tumor specimen that is highlycorrelated with histologic assessments.

DiscussionThe purpose of this study was to develop and validate a multi-

plexed assay forMHCII andTIL gene expression that couldbeusedon FFPE tissue to assess a TNBC patient's risk of recurrence. Theresults of this study demonstrate that performing the MHCIIImmune Activation assay on FFPE tumor specimens using theNanostring nCounter instrument provides accurate measure-ments ofMHCII andTIL gene expression that are highly correlatedwith reduced risk of recurrence in TNBC patients with primarystage I–III breast cancer.

The most likely clinical use of the MHCII Immune Activationassay would be to distinguish TNBC patients who have a very lowrisk of relapse from those who have an average risk of relapse. Wedemonstrate that an Immune Activation Score threshold can beestablished to identify patients who have a very low risk ofrecurrence (Fig. 4) and may not require systemic therapy. Boththe training set and validation cohort in this study includedpatients who did not receive systemic chemotherapy for a varietyof reasons including advanced age, comorbidities, and patientpreference (Supplementary Table S2). Excitingly, we found thatpatients with high Immune Activation Scores who did notreceive systemic chemotherapy did not relapse (SupplementaryFig. S7A). To investigate this preliminary association further, weanalyzed public microarray data from a larger cohort of patientswith primary stage I–III basal-like breast cancer who did notreceive systemic chemotherapy. We found that patients withhigher expression of MHCII and TIL genes had significantlylonger relapse-free survival, even without systemic treatment(Supplementary Fig. S7B). Future clinical studies are warrantedto evaluate whether this assay could be used routinely to identifyTNBC patients who inherently have a good prognosis and cansafely be treated with local therapy alone. The MHCII ImmuneActivation assay enables precision medicine for TNBC patientsand could help reduce the burden of chemotherapy-inducedside effects in TNBC survivors.

Another potential clinical application of the MHCIIImmune Activation assay is predicting response to immuno-therapy. Recent studies have shown that expression of MHCClass II molecules in melanoma cells is associated withimproved response to anti–PD-1 immunotherapy in melano-ma patients (62–64). Data presented at the AmericanSociety of Clinical Oncology 2017 annual meeting from thephase II randomized, controlled, multicenter I-SPY 2 trial(NCT01042379) demonstrated that 60% of newly diagnosedTNBC patients achieved pathologic complete response (pCR)when treated with the immune checkpoint inhibitor pembro-lizumab in combination with standard neoadjuvant chemo-therapy. This was a significant improvement compared withthe 20% of patients who achieved pCR with standard neoad-juvant chemotherapy alone (65). Although this result is prom-ising, it also indicates that 40% of TNBC patients in thepembrolizumab arm did not achieve pCR but were exposedto the significant risks associated with immunotherapy, whichin this trial included autoimmune-mediated adrenal insuffi-ciency, hepatitis, colitis, and hypothyroidism. Future studiesare needed to determine whether the MHCII Immune Activa-tion assay can be used to identify patients that are most likelyto benefit from immunotherapy.

Table 1. Cox regression models of DFS

Univariate MultivariateVariable HR (95% CI) P value HR (95% CI) P value

Training set Immune Activation Score (log10 transformed) 0.1430 (0.03683–0.5555) 0.00496a 0.1688 (0.04039–0.7054) 0.014758a

Stage at diagnosis 2.1227 (1.439–3.131) 0.000147a 2.0310 (1.33617–3.0871) 0.000911a

Age at diagnosis 1.006 (0.9661–1.048) 0.765 1.0363 (0.99081–1.0840) 0.119459Received chemotherapy 0.4879 (0.1752–1.359) 0.17 0.4660 (0.14512–1.4965) 0.199589

Validation cohort Immune Activation Score (log10 transformed) 0.2111 (0.06075–0.7335) 0.01440a 0.1939 (0.05451–0.6896) 0.011280a

Stage at diagnosis 1.628 (1.204–2.201) 0.00154a 1.6363 (1.18309–2.2632) 0.002920a

Age at diagnosis 1.013 (0.9812–1.045) 0.43200 1.0198(0.96152–1.0605) 0.696870Received chemotherapy 0.6166 (0.1986–1.915) 0.40300 0.7696 (0.14986–3.9519) 0.753700

aSignificant P values.






The MHCII Immune Activation assay produces similar mea-surements as histologic assays for MHCII expression and TILcounting (Fig. 5), but provides standardized methodology, alarger dynamic range of measurements, and multiplexed anal-ysis of small specimens. The development of the Prosigna testfor HRþ breast cancer has demonstrated that one key strengthof assays developed on the NanoString nCounter is the abilityto implement them as Laboratory Developed Tests in clinicallaboratory sites across the world while maintaining standard-ized protocols and data analysis. Following demonstration ofits clinical utility, the format of the MHCII Immune Activationassay will enable similar broad adoption as a clinical test forprognosis in TNBC patients, for which there are currently noclinical tests available.

Disclosure of Potential Conflicts of InterestN.L. Henry reports receiving other commercial research support from Pfizer,

AbbVie, Innocrin Pharmaceutical, and H3 Biomedicine. P.S. Bernard hasownership interest (including stock, patents, etc.) in Bioclassifier LLC and

PhenoTx LLC. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: R.L. Stewart, K.L. Updike, P.S. Bernard, K.E. VarleyDevelopment of methodology: R.L. Stewart, P.S. Bernard, K.E. VarleyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.L. Stewart, K.L. Updike, K.E. VarleyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.L. Stewart, K.M. Boucher, P.S. Bernard, K.E. VarleyWriting, review, and/or revision of the manuscript: R.L. Stewart, R.E. Factor,N.L. Henry, K.M. BoucherAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.L. Stewart, K.E. VarleyStudy supervision: K.E. Varley

AcknowledgmentsWe acknowledge the direct financial support for the research reported in this

publication provided by the Huntsman Cancer Foundation and the Women'sCancer Disease Oriented Team at Huntsman Cancer Institute. The projectdescribed was also supported by the NIH National Center for Advancing

Figure 5.

Comparison of the MHCII ImmuneActivation assay with IHC andhistologic TIL counting. A, IHCanalysis of MHCII expression inpatients with high, intermediate,and low Immune Activation Scores.B, The TIL Score calculated from TILgene expression using the MHCIIImmune Activation assay iscorrelated with histologicassessment of stromal TILpercentage.

Stewart et al.





Translational Sciences through grant number KL2TR001996 (R.L. Stewart).Research reported in this publication utilized the University of Utah HuntsmanCancer Institute Biorepository and Molecular Pathology Shared Resource andthe High-Throughput Genomics Shared Resource, which are supported by theNCI of the NIH under Award Number P30CA042014. This research was alsosupported by the Biospecimen Procurement and Translational Pathology andOncogenomics Shared Resource Facilities of the University of KentuckyMarkeyCancer Center (P30CA177558). The content is solely the responsibility of theauthors and does not necessarily represent the official views of the NIH. Specialthanks to Sheryl Tripp at ARUP Laboratories for her histologic expertise, and

special thanks to Darah Johnson, S. Emily Bachert, and Donna Wall at theUniversity of Kentucky for their technical assistance.

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 September 25, 2018; revised February 21, 2019; accepted April 29,2019; published first May 2, 2019.

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2019;79:3466-3478. Published OnlineFirst May 2, 2019.Cancer Res Rachel L. Stewart, Katherine L. Updike, Rachel E. Factor, et al. Triple-Negative Breast CancerA Multigene Assay Determines Risk of Recurrence in Patients with

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