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Translational Science
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.
CancerResearch
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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 (
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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
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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.
Stewart et al.
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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
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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.
Stewart et al.
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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.
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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.
Stewart et al.
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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.
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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.
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-
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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Cancer Res; 79(13) July 1, 2019 Cancer Research3478
Stewart et al.
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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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