Research

Brief Report

Association of Depressed Anti-HER2 T-Helper Type 1

Response With Recurrence in Patients With Completely
Treated HER2-Positive Breast Cancer
Role for Immune Monitoring
Jashodeep Datta, MD; Megan Fracol, MD; Matthew T. McMillan, BA; Erik Berk, PhD; Shuwen Xu, MD;
Noah Goodman, MPH; David A. Lewis, BA; Angela DeMichele, MD, MSCE; Brian J. Czerniecki, MD, PhD

Supplemental content at
IMPORTANCE There is a paucity of immune signatures identifying patients with human jamaoncology.com
epidermal growth factor receptor 2 (HER2)-positive invasive breast cancer (IBC) at risk for
treatment failure following trastuzumab and chemotherapy.

OBJECTIVE To determine whether circulating anti-HER2 CD4-positive (CD4+) T-helper type 1

(Th1) immunity correlates with recurrence in patients with completely treated HER2-positive
IBC.

DESIGN, SETTING, AND PARTICIPANTS Hypothesis-generating exploratory translational

analysis at a tertiary care referral center of patients with completely treated HER2-positive
IBC with median (interquartile range) follow-up of 44 (31) months. Anti-HER2 Th1 responses
were examined using peripheral blood mononuclear cells pulsed with 6 HER2-derived class
II–promiscuous peptides via interferon-γ (IFN-γ) enzyme-linked immunospot assay.

MAIN OUTCOMES AND MEASURES T-helper type 1 response metrics were anti-HER2
responsivity, repertoire (number of reactive peptides), and cumulative response across 6
peptides (spot-forming cells [SFCs]/106 cells). Anti-HER2 Th1 responses in treatment-naive
patients (used as an immunologic baseline) were compared with those in patients completing
trastuzumab and chemotherapy; in the latter group, analyses were stratified by recurrence
status. Recurrence was defined as any locoregional or distant breast event, or both. Cox
regression analysis estimated the instantaneous hazard of recurrence (ie, disease-free
survival [DFS]) stratified by anti-HER2 Th1 responsivity.

RESULTS In 95 women with HER2-positive IBC (median [range] age, 49 [24-85] years; 22
treatment-naive, 73 treated with trastuzumab and chemotherapy), depressed anti-HER2 Th1
responsivity (recurrence, 2 of 25 [8%], vs nonrecurrence, 40 of 48 [83%]; P < .001), mean (SD) Author Affiliations: Division of
repertoire (0.1 [0.1] vs 1.5 [0.2]; P < .001), and mean (SD) cumulative response (14.8 [2.0] vs Endocrine and Oncologic Surgery,
80.2 [11.0] SFCs/106 cells; P < .001) were observed in patients incurring recurrence (n = 25) Department of Surgery, Hospital of
the University of Pennsylvania,
compared with patients without recurrence (n = 48). After controlling for confounding, Philadelphia (Datta, Fracol, McMillan,
anti-HER2 Th1 responsivity remained independently associated with recurrence (P < .001). Berk, Xu, Czerniecki); Division of
This immune disparity was mediated by anti-HER2 CD4+T-bet+IFN-γ+ (Th1)—not Medical Oncology, Department of
Medicine, Hospital of the University
CD4+GATA-3+IFN-γ+ (Th2) or CD4+CD25+FoxP3+ (Treg)—phenotypes, and not attributable to
of Pennsylvania, Philadelphia
immune incompetence. When stratifying trastuzumab plus chemotherapy-treated patients by (Goodman, Lewis, DeMichele);
Th1 responsivity, Th1-nonresponsive patients demonstrated a worse DFS (median, 47 vs 113 Department of Epidemiology and
months; P < .001) compared with Th1-responsive patients (hazard ratio, 16.9 [95% CI, 3.9-71.4]; Biostatistics, Hospital of the
University of Pennsylvania,
P < .001). Philadelphia (DeMichele); Rena
Rowen Breast Center, Hospital of the
CONCLUSIONS AND RELEVANCE Depressed anti-HER2 Th1 response is a novel immune University of Pennsylvania,
Philadelphia (Czerniecki).
correlate to recurrence in patients with completely treated HER2-positive IBC. These data
underscore a role for immune monitoring in patients with HER2-positive IBC to identify Corresponding Author: Brian J.
Czerniecki, MD, PhD, Department of
vulnerable populations at risk of treatment failure. Surgery, University of Pennsylvania
Perelman School of Medicine, Rena
Rowen Breast Center, 3400 Civic
JAMA Oncol. doi:10.1001/jamaoncol.2015.5482 Center Dr, Philadelphia, PA 19104
Published online December 30, 2015. (brian.czerniecki@uphs.upenn.edu).

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 Research Brief Report
A
lthough human epidermal growth factor receptor 2
(HER2)-targeted therapies, in combination with chemo-
therapy, have dramatically improved survival in HER2-
positive breast cancer (BC),1 a substantial proportion of these
patients develop resistance and ultimately experience recur-
rence.2 Immune signatures identifying patients at risk for such
treatment failure are lacking. We recently demonstrated a pro-
gressive loss in anti-HER2 CD4-positive (CD4+) T-helper type 1
(Th1) immunity across a tumorigenesis continuum in HER2-
positive BC, extending from healthy donors, through patients
with HER2-positive ductal carcinoma in situ, and ultimately pa-
tients with HER2-positive invasive BC (IBC).3 Additionally, anti-
HER2 Th1 response emerged as a novel immune correlate to
pathologic response following neoadjuvant trastuzumab plus
chemotherapy in HER2-positive BC.4 In light of these observa-
tions, we hypothesized that anti-HER2 Th1 immunity may be
associated with disease recurrence. In an exploratory cohort, we
examined differences in circulating anti-HER2 Th1 immunity be-
tween recurrent and disease-free HER2-positive IBC patients in
order to identify immune correlates to recurrence.
Methods
Study Design
On approval by the institutional review board of the Univer-
sity of Pennsylvania, 95 patients with HER2-positive BC were
recruited prospectively in a nonbiased fashion after written in-
formed consent was obtained. Eligible patients had histologi-
cally confirmed IBC, ERBB2 (formerly HER2 or HER2/neu) over-
expression (3+ [n = 82] or 2+/fluorescence in situ hybridization
positive [n = 13]), and were not receiving immunosuppres-
sive medications. Anti-HER2 Th1 responses in treatment-
naive (no definitive therapy at enrollment) patients with stage
I to III HER2-positive IBC (n = 22) were compared with Th1 re-
sponses in patients with stage I to IV HER2-positive IBC (n = 73)
who had completed trastuzumab plus chemotherapy treat-
ment—either neoadjuvant (n = 37; immune responses from this
cohort have been reported previously4) or adjuvant trastu-
zumab plus chemotherapy (n = 36) plus definitive surgery
(schedules and dosing of trastuzumab plus chemotherapy regi-
mens are presented in the eMethods in the Supplement). In
trastuzumab plus chemotherapy–treated patients, analyses
were stratified by recurrence status. Although recurrence sta-
tus was known at study enrollment, evaluation and analysis
of anti-HER2 Th1 responses was blinded to this information.
Human epidermal growth factor receptor 2–positive–
confirmed recurrences were defined as any locoregional or
distant breast event. Patients incurring recurrence were
enrolled prior to initiation of second-line chemotherapy,
HER2-targeted therapy, or experimental (eg, HER2–pulsed
dendritic cell vaccination) protocols. Patients without recur-
rence were eligible for analysis only if disease-free interval
was 24 months at minimum (eFigure 1 in the Supplement).
Immune Response Detection
Anti-HER2 responses were examined in unexpanded periph-
eral blood mononuclear cells (PBMCs) pulsed ex vivo with 6 vali-
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Anti-HER2 T-Helper Type 1 Response and Breast Cancer Recurrence
At a Glance
• We investigated whether circulating anti–human epidermal
growth factor receptor 2 (HER2) CD4+ T-helper type 1 (Th1)
immunity correlates with disease recurrence (locoregional,
distant, or both) in HER2-positive breast cancer patients who
have completed HER2-targeted (ie, trastuzumab) therapy.
• Anti-HER2 T-cell responses were depressed in patients incurring
recurrence compared with disease-free patients (P < .001).
• HER2 Th1 responsivity remained independently associated with
recurrence (P < .001), and Th1-nonresponsive patients
demonstrated a worse disease-free survival (median, 47 vs 113
months; P < .001) vs Th1-responsive patients.
• Immune monitoring in completely treated HER2-positive
patients may identify populations at risk of clinicopathologic
failure.
dated HER2-derived major histocompatibility complex class II–
promiscuous peptides,5 by measuring interferon-γ (IFN-γ),
interleukin-4 (IL-4), or IL-10 production via enzyme-linked im-
munospot assay, as previously described (eMethods in the
Supplement).3,4,6 Specifically, PBMCs were incubated with
either HER2 peptides (4 μg; Genscript), media alone (unstimu-
lated control), or positive control (anti–human CD3/CD28 an-
tibodies [0.5 μg/mL; BD Pharmingen]) at 37°C for 36 to 48 hours.
In evaluable patients, Th1 responses to 1:100-diluted re-
call stimuli Candida albicans (Allermed Laboratories) and teta-
nus toxoid (Santa Cruz Biotechnology) whole proteins were
examined.3 Anti-HER2 Th1 reactivity was determined using
empirical methodology as described previously.3,4 Positive/
reactive response to a HER2 peptide was defined as at least 20
spot-forming cells (SFCs)/2 × 105 PBMCs in experimental wells
after subtraction of unstimulated background. Three metrics
of immune response were measured: (1) responsivity (propor-
tion of patients responding to ≥1 of 6 peptides), (2) repertoire
(mean number of reactive peptides), and (3) cumulative re-
sponse across 6 peptides (SFCs/106 cells).
Flow cytometry, the functional contribution of Th1 vs Th2
subtypes, and statistical analysis are described in the eMethods
in the Supplement.
Statistical Analysis
Descriptive statistics summarized distributions of patient char-
acteristics and immune response variables. Comparisons be-
tween nonrecurrent and recurrent HER2-positive IBC co-
horts were performed as indicated: (1) 2-group/univariate
testing: unpaired Student t test (parametric continuous), Wil-
coxon rank-sum test (nonparametric continuous), and χ2 tests
(categorical); (2) more than 2–group testing: 1-way analysis of
variance with post hoc Bonferroni testing. Variables with P < .1
on univariate testing were entered into a forward, stepwise
multivariable logistic regression model (P < .05 for entry) to
determine independent correlates to recurrence (binary out-
come yes/no). Missing data for postneoadjuvant pathologic re-
sponse status (n = 36) and lymphovascular invasion (n = 12)
were imputed using the Markov chain Monte Carlo method.
Five imputation data sets were created and 5 sets of analyses
were combined per the formula of Rubin.
jamaoncology.com
 Anti-HER2 T-Helper Type 1 Response and Breast Cancer Recurrence
Univariate DFS estimates were examined by Kaplan-
Meier methodology, stratifying by anti-HER2 Th1 responsiv-
ity and other covariates. Observations of patients without re-
currence (minimum 24 month follow-up) were censored at last
known follow-up. To analyze the instantaneous hazard of all
variables and control for varied follow-up, Cox proportional
hazards modeling was performed. The assumptions of the Cox
model were assessed, including interactions and proportion-
ality of hazards over time. P < .05 was considered statisti-
cally significant. All tests were 2 sided. Analyses were per-
formed using SPSS, version 22 (IBM Corp).
Results
Patient Characteristics
Demographic and tumor-related characteristics of the overall
cohort (n = 95, all female) are detailed in eTable 1 in the Supple-
ment. In trastuzumab plus chemotherapy–treated patients
(n = 73), median (range) age was 49 (24-85) years; a majority
of patients had estrogen receptor–positive/progesterone re-
ceptor–positive tumors (43 [59%]) and presented with locally
advanced/node-positive disease (clinical stage I, 7 [10%]; stage
II, 35 [48%]; stage III, 31 [42%]). Neoadjuvant trastuzumab plus
chemotherapy was administered in 37 (51%) patients, and doxo-
rubicin-cyclophosphamide-paclitaxel-trastuzumab was the
commonly used regimen (49 [67%]) (eMethods in the Supple-
ment). Simple or modified radical mastectomy was per-
formed most frequently (39 [53%]).
Twenty-five of 73 (34%) patients incurred either locore-
gional recurrence, distant recurrence, or both (eTable 2 in
the Supplement); Th1 responses in a majority (21 [84%]) of
these patients were determined at the time of diagnosis of
recurrence.
Depression of Anti-HER2 Th1 Responses in Patients
With HER2-Positive IBC Incurring Recurrence
Anti-HER2 Th1 responsivity, repertoire, and cumulative re-
sponses were compared between treatment-naive (n = 22),
trastuzumab plus chemotherapy–treated nonrecurrent (n = 48),
and trastuzumab plus chemotherapy–treated patients with re-
current disease (n = 25). Using anti-HER2 Th1 responses from
treatment-naive patients as an immunologic “baseline,” sig-
nificantly depressed anti-HER2 Th1 responsivity (treatment-
naive, 8 [36%], vs recurrence, 2 [8%], vs nonrecurrence, 40
[83%]; P < .001), mean (SD) repertoire (0.6 [0.2] vs 0.1 [0.1] vs
1.5 [0.2]; P < .001), and mean (SD) cumulative response
(32.8 [4.7] vs 14.8 [2.0] vs 80.2 [11.0] SFCs/106 cells; P < .001)
were observed in patients incurring recurrence compared with
disease-free patients (Figure 1A).
Interferon-γ+ responses to anti-CD3/anti-CD28 stimula-
tion (P = .57) (Figure 1B) or tetanus (P = .41) and Candida
(P = .74) (eFigure 2 in the Supplement) did not differ signifi-
cantly between the nonrecurrence and recurrence cohorts, sug-
gesting that the observed anti-HER2 Th1 disparity is not at-
tributable to immune incompetence or host-level T-cell anergy
in patients with recurrent disease. Decreased mean (SEM) pro-
portions of HER2-specific CD4+T-bet+IFN-γ+ (0.25 [0.1%] vs
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Brief Report Research
0.02 [0.01%]; P = .04), but not CD4 + GATA-3 + IFN-γ + or
CD4+GATA-3+IFN-γ-, phenotypes were observed in the recur-
rence compared with nonrecurrence cohort, respectively
(Figure 1C).
Neither HER2-stimulated IL-4+ measures of Th2 func-
tion (eFigure 3A in the Supplement), HER2-stimulated IL-10+
measures of Treg function (eFigure 3B in the Supplement), nor
relative proportions of circulating Treg (ie, CD4+CD25+Foxp3+)
phenotypes (Figure 1D) differed between nonrecurrence and
recurrence cohorts.
Association of Anti-HER2 Th1 Responsivity
With Disease-Free Survival
Next, independence of the association between anti-HER2 Th1
response and recurrence was examined. On univariate test-
ing, recurrence and nonrecurrence cohorts did not differ by
age, menopausal status, race, body mass index, comorbidity,
stage, estrogen receptor–positive/progesterone receptor–
positive status, lymphovascular invasion, nuclear grade, or
need for mastectomy. However, recurrence was more fre-
quent among patients receiving adjuvant trastuzumab plus
chemotherapy (P = .02) and those with residual disease fol-
lowing neoadjuvant therapy (P = .006). When controlling for
these variables via multivariable regression, anti-HER2 Th1 re-
sponsivity (P < .001), but not trastuzumab plus chemo-
therapy sequence (P = .14) or pathologic response (P = .76), re-
mained independently associated with recurrence (eTable 3
in the Supplement).
Stratifying the analytic cohort by anti-HER2 Th1 respon-
sivity at a median (interquartile range) follow-up of 44 (28-
59) months, Th1-nonresponsive patients demonstrated a sig-
nificantly worse disease-free survival (median, 47 vs 113
months; P < .001) compared with Th1-responsive patients; this
association was corroborated by Cox modeling (hazard ratio
for Th1 nonresponsive cohort, 16.9 [95% CI, 3.9-71.4]; P < .001)
(Figure 2).
Discussion
In this hypothesis-generating exploratory analysis, circulat-
ing anti-HER2 CD4 + Th1 response emerges as a novel
immune correlate to breast cancer recurrence in patients
with completely treated HER2-positive IBC. While not attrib-
utable to immune incompetence, depressed anti-HER2
T-cell responses in patients with recurrent disease were
driven predominantly by Th1 phenotypes. When relevant
clinicopathologic factors were controlled for, anti-HER2 Th1
responsivity was independently associated with recurrence;
on risk-adjusted analysis, Th1-nonresponsive patients dem-
onstrated worse disease-free survival compared with Th1-
responsive patients. To our knowledge, this is the first dem-
onstration of an association between disease recurrence and
a host-level immune correlate specific to an oncodriver in
breast tumorigenesis.
These data are intriguing in the light of evidence suggest-
ing that benefit from trastuzumab therapy—both in the
adjuvant7 and neoadjuvant8 setting—may be restricted to im-
(Reprinted) JAMA Oncology Published online December 30, 2015 E3
 Research Brief Report Anti-HER2 T-Helper Type 1 Response and Breast Cancer Recurrence

Figure 1. Comparison of Disease-Free Patients vs Patients With Recurrence

A T-cell response variations P < .001 B Immune competence in PBMCs (anti-CD3/anti-CD28)
100 P < .001 2000 CD3/CD28
P = .57

Mean Total SFCs/2 × 105 Cells

Anti-HER2 Responsivity, %

80

1500
60 P < .05

40
1000

20

0
Treatment Naive Recurrence Nonrecurrence Nonrecurrence Recurrence
(n = 22) (n = 25) (n = 48) (n = 48) (n = 25)
6 C Contributions of Th1 vs Th2 phenotypes
P < .001 0.4 P = .83
5 Nonrecurrence
Mean No. of Reactive Peptides

P < .001 P =.04

Recurrence
4 P >.99
0.3

CD4+ PBMCs, %
3 P = NS
0.2
2

1 0.1

0
0
Treatment Naive Recurrence Nonrecurrence T-bet+ GATA-3+ GATA-3+ T-bet+ GATA-3+ GATA-3+
(n = 22) (n = 25) (n = 48) IFN-γ+ IFN-γ+ IFN-γ- IFN-γ+ IFN-γ+ IFN-γ-
P < .001

P < .001 D Proportions of Treg

400 5
Nonrecurrence
(n = 8)
Recurrence
4
Mean Total SFCs/106 Cells

300 (n = 6)

3
PBMC, %

200
P = .74
P = NS 2

100

0
0
Treatment Naive Recurrence Nonrecurrence CD4+CD25+ FoxP3+
(n = 22) (n = 25) (n = 48)

A, Interferon-γ–positive (IFN-γ+) anti-HER2 CD4+ T-cell response variations
between human epidermal growth factor receptor 2 (HER2)-positive invasive
breast cancer patient cohorts (treatment-naive [n = 22], recurrence [n = 25],
and nonrecurrence [n = 48]), stratified by anti-HER2 responsivity, response
repertoire (mean number of reactive peptides), and cumulative response (mean
total spot-forming cells [SFCs]/106 cells). Bars indicate means in percent; error
bars, SEM. One-way analysis of variance P values are shown alongside as
calculated by means of post hoc Bonferroni testing. NS indicates nonsignificant.
B, Peripheral blood mononuclear cells (PBMCs) from nonrecurrence and
recurrence cohorts did not differ significantly in immune competence—
measured by IFN-γ production to anti-CD3/anti-CD28 stimulus by
enzyme-linked immunospot assay. The horizontal line in the middle of each box
indicates the median IFN-γ SFCs/2 x 105 cells, while the top and bottom borders
of the box mark the 75th and 25th percentiles, respectively. The whiskers above
and below the box mark the 90th and 10th percentiles. C, Relative
contributions of Th1 (T-bet+IFN-γ+) vs Th2 (GATA-3+IFN-γ+) phenotypes to
HER2 peptide-specific IFN-γ+ cells in nonrecurrence and recurrence cohorts’
PBMCs. Bars indicate means in percent; error bars, SEM. D, Relative proportions
of Treg (CD4+CD25+FoxP3+) by flow cytometry. Bars indicate means in percent;
error bars, SEM. NS indicates nonsignificant.

mune-enriched tumors, with Th1 genes IFN-γ and tumor ne- tients with completely treated HER2-positive BC for real-
crosis factor imparting a particularly dominant role.7 Alto- time fluctuations in anti-HER2 Th1 immunity may reveal vul-
gether, it appears that absent tumor-level Th1 gene expression, nerable populations at risk of relapse and identify critical
as well as deficient circulating anti-HER2 Th1 immunity, may opportunities for therapeutic intervention, such as anti-
presage failure of HER2-targeted therapy. Monitoring pa- HER2 Th1-directed immune interventions.4

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 Anti-HER2 T-Helper Type 1 Response and Breast Cancer Recurrence Brief Report Research

Since immune responses were examined at the time of

Figure 2. Cox Proportional Hazards Modeling of Disease-Free Survival
in Patients With Completely Treated Human Epidermal Growth Factor recurrence, it remains unclear whether anti-HER2 Th1 immu-
Receptor 2 (HER2)-Positive Breast Cancer, Stratified by Anti-HER2 nity in these patients remained suppressed following comple-
T-Helper Type 1 (Th1) Responsivity. tion of index trastuzumab therapy or declined contemporane-
ously with development of recurrence; longitudinal
1.0 surveillance of anti-HER2 Th1 responses is required. Other
limitations warrant emphasis. These findings should be inter-
preted as hypothesis generating and warrant large-scale vali-
0.8 dation; this study did not address other HER2-targeted agents
(eg, lapatinib, pertuzumab); the relative preponderance of
patients with locally advanced disease in our cohort may
Disease-Free Survival

0.6
diminish applicability in early-stage disease; and finally, while
the case for anti-HER2 Th1 immune monitoring is compelling,
our study is unable to establish numerical thresholds for such
0.4
monitoring or correlations between the depth of immune
depression and recrudescent tumor burden.

0.2 Anti-HER2 Th1

Responsivity
Hazard ratio, 16.9
No
(95% CI, 3.9-71.4)
Yes
P < .001 Conclusions
0
0 20 40 60 80 100 120 Depressed anti-HER2 Th1 response is a novel immune corre-
Time, mo late to recurrence in patients with completely treated HER2-
positive IBC. These data underscore a role for immune moni-
Non-Th1–responsive patients demonstrate worse risk-adjusted disease-free toring in patients with HER2-positive IBC to identify vulnerable
survival relative to Th1-responsive patients.
populations at risk of treatment failure.

ARTICLE INFORMATION Pennies in Action (http://www.penniesinaction.org), correlate to pathologic response following

Accepted for Publication: October 30, 2015
Published Online: December 30, 2015.
doi:10.1001/jamaoncol.2015.5482.
Author Contributions: Drs Datta and Czerniecki
had full access to all of the data in the study and
take responsibility for the integrity of the data and
the accuracy of the data analysis.
Study concept and design: Datta, McMillan,
Goodman, DeMichele, Czerniecki.
Acquisition, analysis, or interpretation of data:
Datta, Fracol, McMillan, Berk, Xu, Lewis, DeMichele,
Czerniecki.
Drafting of the manuscript: Datta, McMillan,
Goodman, Czerniecki.
Critical revision of the manuscript for important
intellectual content: Datta, Fracol, McMillan, Berk,
Xu, Lewis, DeMichele, Czerniecki.
Statistical analysis: Datta, Fracol, McMillan, Lewis.
Obtained funding: Datta, Czerniecki.
Administrative, technical, or material support: Datta,
Fracol, Berk, Xu, Goodman, Lewis, DeMichele,
Czerniecki.
Study supervision: DeMichele, Czerniecki.
Conflict of Interest Disclosures: None reported.
Funding/Support: This work was supported by
National Institutes of Health grant R01 CA096997,
and a University of Pennsylvania Abramson Cancer
Center Breast Translational Center of Excellence
grant.
Role of the Funder/Sponsor: The funding agencies
had no role in the design and conduct of the study;
collection, management, analysis, and
interpretation of the data; preparation, review, or
approval of the manuscript; and decision to submit
the manuscript for publication.
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