cancers
Article
ERBB2 mRNA Expression and Response to
Ado-Trastuzumab Emtansine (T-DM1) in
HER2-Positive Breast Cancer
Gaia Griguolo 1,2,† , Fara Brasó-Maristany 3,4,† , Blanca González-Farré 4,5 , Tomás Pascual 3,4,6 ,
Núria Chic 3,4 , Tamara Saurí 3,4 , Ronald Kates 7 , Oleg Gluz 7,8,9 , Débora Martínez 3,4 , Laia Paré 6 ,
Vassilena Tsvetkova 2 , David Pesantez 3,4 , Maria Vidal 3,4,6 , Barbara Adamo 3,4 ,
Montserrat Muñoz 3,4,6 , Patricia Galván 3,4 , Laura Barberá 3,4 , Miriam Cuatrecasas 5 ,
Mathias Christgen 10 , Hans Kreipe 10 , Inés Monge-Escartín 11 , Patricia Villagrasa 6 ,
Dolors Soy 11 , Tommaso Giarratano 1 , Maria Vittoria Dieci 1,2 , Pierfranco Conte 1,2 ,
Nadia Harbeck 12 , Valentina Guarneri 1,2 and Aleix Prat 3,4,6, *
1
2
3
4
5
6
7
8
9
10
11
12
*
†
Division of Medical Oncology 2, Istituto Oncologico Veneto IOV—IRCCS, 35128 Padova, Italy;
gaia.griguolo@iov.veneto.it (G.G.); tommaso.giarratano@iov.veneto.it (T.G.);
mariavittoria.dieci@unipd.it (M.V.D.); pierfranco.conte@unipd.it (P.C.); valentina.guarneri@unipd.it (V.G.)
Department of Surgery, Oncology and Gastroenterology, University of Padova, 35124 Padova, Italy;
vassilena.tsvetkova@gmail.com
Department of Medical Oncology, Hospital Clínic de Barcelona, 08036 Barcelona, Spain;
fbraso@clinic.cat (F.B.-M.); topascual@clinic.cat (T.P.); chic@clinic.cat (N.C.); sauri@clinic.cat (T.S.);
demartinez@clinic.cat (D.M.); Pesantez@clinic.cat (D.P.); MJVIDAL@clinic.cat (M.V.);
ADAMO@clinic.cat (B.A.); MMUNOZ@clinic.cat (M.M.); galvan@clinic.cat (P.G.);
LBARBERA@clinic.cat (L.B.)
Translational Genomics and Targeted Therapeutics in Solid Tumors, August Pi i Sunyer Biomedical Research
Institute (IDIBAPS), 08036 Barcelona, Spain; MBGONZAL@clinic.cat
Department of Pathology, Hospital Clínic de Barcelona, 08036 Barcelona, Spain; MCUATREC@clinic.cat
SOLTI breast cancer cooperative group, 08008 Barcelona, Spain; Laia.pare@gruposolti.org (L.P.);
patricia.villagrasa@gruposolti.org (P.V.)
The West German Study Group, 41061 Mönchengladbach, Germany; ronald.kates@t-online.de (R.K.);
oleg.gluz@wsg-online.com (O.G.)
Ev. Hospital Bethesda, Breast Center Niederrhein, 41061 Mönchengladbach, Germany
University Clinics Cologne, 50937 Cologne, Germany
Medical School Hannover, Institute of Pathology, 30625 Hannover, Germany;
christgen.matthias@mh-hannover.de (M.C.); Kreipe.Hans@mh-hannover.de (H.K.)
Pharmacy Department, Division of Medicines, Hospital Clínic de Barcelona, 08036 Barcelona, Spain;
monge@clinic.cat (I.M.-E.); DSOY@clinic.cat (D.S.)
Breast Center, Department of Gynecology and Obstetrics, University of Munich (LMU) and CCCLMU,
80337 Munich, Germany; Nadia.Harbeck@med.uni-muenchen.de
Correspondence: alprat@clinic.cat
These authors contributed equally to this work.
Received: 13 June 2020; Accepted: 8 July 2020; Published: 14 July 2020
Abstract: Trastuzumab emtansine (T-DM1) is approved for the treatment of human epidermal growth
factor receptor 2 (HER2)-positive (HER2+) metastatic breast cancer (BC) and for residual disease after
neoadjuvant therapy; however, not all patients benefit. Here, we hypothesized that the heterogeneity
in the response seen in patients is partly explained by the levels of human epidermal growth factor
receptor 2 gene (ERBB2) mRNA. We analyzed ERBB2 expression using a clinically applicable assay
in formalin-fixed paraffin-embedded (FFPE) tumors (primary or metastatic) from a retrospective
series of 77 patients with advanced HER2+ BC treated with T-DM1. The association of ERBB2 levels
and response was further validated in 161 baseline tumors from the West German Study (WGS)
Group ADAPT phase II trial exploring neoadjuvant T-DM1 and 9 in vitro BC cell lines. Finally,
Cancers 2020, 12, 1902; doi:10.3390/cancers12071902
www.mdpi.com/journal/cancers
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ERBB2 expression was explored in 392 BCs from an in-house dataset, 368 primary BCs from The
Cancer Genome Atlas (TCGA) dataset and 10,071 tumors representing 33 cancer types from the
PanCancer TCGA dataset. High ERBB2 mRNA was found associated with better response and
progression-free survival in the metastatic setting and higher rates of pathological complete response
in the neoadjuvant setting. ERBB2 expression also correlated with in vitro response to T-DM1.
Finally, our assay identified 0.20–8.41% of tumors across 15 cancer types as ERBB2-high, including
gastric and esophagus adenocarcinomas, urothelial carcinoma, cervical squamous carcinoma and
pancreatic cancer. In particular, we identified high ERBB2 mRNA in a patient with HER2+ advanced
gastric cancer who achieved a long-lasting partial response to T-DM1. Our study demonstrates that
the heterogeneity in response to T-DM1 is partly explained by ERBB2 levels and provides a clinically
applicable assay to be tested in future clinical trials of breast cancer and other cancer types.
Keywords: ERBB2 mRNA; HER2-positive breast cancer; T-DM1; antibody-drug conjugates
1. Introduction
Trastuzumab emtansine (T-DM1) is an antibody–drug conjugate (ADC) linking the anti-HER2
(human epidermal growth factor receptor 2) monoclonal antibody trastuzumab to a microtubule
inhibitor, DM1. T-DM1 is approved in several countries as single-agent treatment for HER2+ metastatic
breast cancer (BC) patients previously treated with trastuzumab and a taxane. In the phase III
randomized EMILIA trial, T-DM1 was compared with capecitabine plus lapatinib in previously treated
(trastuzumab and taxane) HER2+ metastatic BC patients [1]. In the phase III randomized TH3RESA
trial, enrolling patients previously treated with trastuzumab and lapatinib in the advanced setting,
T-DM1 was compared with treatment of physician’s choice [2]. Treatment with T-DM1 was associated
with a significant improvement in both progression-free survival (PFS) and overall survival (OS)
in both trials. Recently, trastuzumab emtansine (T-DM1) has also been approved (Food and Drug
Administration and European Medicines Agency) for the treatment of residual invasive disease after
neoadjuvant treatment for HER2+ BC. In fact, the phase III randomized KATHERINE trial, which
enrolled HER2+ BC who were found to have residual invasive disease (breast or axilla) after receiving
neoadjuvant therapy containing at least a taxane and trastuzumab, demonstrated a clear improvement
in invasive disease-free survival for patients who were switched to T-DM1 instead of continuing
trastuzumab [3].
HER2-positivity is currently defined by semi-quantitative methods such as immunohistochemistry
(IHC) and in situ hybridization (ISH). This definition was originally designed to predict benefit from
trastuzumab in advanced and adjuvant trials [4,5], and has remained the definition for the development
of the rest of anti-HER2 therapies, including T-DM1. However, the classic definition of HER2-positive
breast cancer has been recently been challenged. For example, the presence of HER2 intratumor
heterogeneity plays a significant role in modulating response to anti-HER2 treatments and is associated
with worse patient outcomes, in terms of shorter disease free survival and overall survival [6–8],
an aspect scarcely accounted for by semi-quantitative evaluations; at the same time, new HER2-targeted
ADCs approved [9] or currently under investigation in phase III trials for HER2+ advanced BC have
been reported to be active in HER2-low BC (defined as IHC 1+ or 2+/non amplified) [10–12], thus raising
the question if different definitions of HER2-positivity should be adopted for each HER2-targeted agent.
Here, we hypothesized that quantitative measurements of HER2, such as ERBB2 mRNA expression,
might further help better identify within HER2+ metastatic BC patients those who will benefit
from T-DM1.
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2. Results
2.1. ERBB2 mRNA in Advanced HER2+ BC Treated with T-DM1
Seventy-seven consecutive patients diagnosed with HER2+ advanced BC and treated with T-DM1
at Hospital Clínic of Barcelona (HCB) and Istituto Oncologico Veneto (IOV) in Padova were evaluated.
Demographic and disease characteristics of these patients are presented in Table 1. Briefly, all patients
were pre-treated with trastuzumab in the (neo)adjuvant or metastatic setting and most had received
at least 1 line of HER2-targeted treatment for metastatic disease (median 1, range 0–4). In addition,
twenty-eight patients (36%) presented brain metastases at time of initiation of T-DM1. Regarding
T-DM1 efficacy, overall response rate (ORR) was 47% (6 complete and 30 partial responses) and median
progression-free survival (PFS) was 5.3 months (95% CI 4.4–10.7).
Table 1. Patient characteristics.
Characteristics
n = 77
Median age at BC diagnosis, years (range)
Median age at start of T-DM1, years (range)
Histology: Ductal
Lobular/other
NA
Histologic Grade: G1
G2
G3
NA
Hormone-receptor: positive
negative
HER2 IHC status: IHC 0
IHC 1+
HC 2+
IHC 3+
NA *
HER2 ISH status in HER2 IHC 2+ cases: Amplified
Not evaluable *
HER2 ISH status in HER2 IHC 0/1+ cases: Amplified
Non-amplified *
Not available *
Previous (neo)adjuvant treatment
Median number previous lines HER2-targeted
treatment for metastatic disease (range)
Previously received:
Pertuzumab-trastuzumab
Trastuzumab
Lapatinib
Visceral metastases at start of T-DM1
Brain metastases at start of T-DM1
Concomitant endocrine treatment during T-DM1
49 (27–88)
51 (35–93)
71 (92%)
5 (6%)
1 (1%)
2 (3%)
10 (13%)
28 (36%)
37 (48%)
46 (60%)
31 (40%)
5 (6%)
3 (4%)
16 (21%)
50 (65%)
3 (4%)
15 (94%)
1 (6%)
3 (38%)
1 (12%)
4 (50%)
44 (57%)
1 (0–4)
31 (40%)
41 (53%)
14 (24%)
66 (86%)
28 (36%)
17 (22%)
* These cases were confirmed to be HER2-positive in other tumor samples and treated with T-DM1 according to
clinical practice.
ERBB2 mRNA expression was assessed in 77 tumor samples from patients with advanced HER2+
BC treated with T-DM1. After determination of an ERBB2 mRNA cutoff predictive of response to
T-DM1, several cohorts were evaluated. On one hand, the ERBB2 mRNA assay was validated in 161
patients recruited in the WSG-ADAPT phase II neoadjuvant trial of T-DM1. On the other, ERBB2
mRNA expression and response was evaluated in 9 in vitro BC cell lines. Finally, ERBB2 mRNA
expression was explored in 392 BCs from an in-house dataset, 368 primary BCs from the TCGA BC
dataset, and 10,071 tumors representing 33 cancer types from the PanCancer TCGA dataset (Figure 1).
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Figure 1. Workflow of the study.
A large range of ERBB2 mRNA expression was observed (log2 median 2.98; interquantile range
1.60–3.91). As expected, the expression of ERBB2 varied according to HER2 IHC expression (0, 1+, 2+
and 3+) (Figure S1). Compared to 0–2+, the expression in 3+ tumor samples was increased by 5.7-fold.
Of note, although all patients were classified as HER2+ tumors by guidelines [4], based on clinical
history and previous assessment of HER2 status on tumor samples, and were eligible for treatment
with T-DM1, 8 of the 74 samples that were re-analyzed for HER2 had an IHC HER2 result of 0 (n = 5)
or 1+ (n = 3). Four of these 8 samples were tested for HER2 amplification by ISH, and HER2 was found
amplified in 3 cases and non-amplified in 1 sample.
The clinicopathological variables associated with response (i.e., partial and complete) to T-DM1
were: negative hormone-receptor status, lower number of prior lines of HER2-targeted therapy in
the metastatic setting, higher HER2 IHC expression (i.e., 3+ vs. 0–2+) and higher ERBB2 mRNA as a
continuous variable (Table 2). The overall response rate in HER2 3+ was 62.0% compared to 20.8% in
the 0–2+ group (odds ratio = 1.84, 95% confidence interval [CI] 1.26–2.69, p = 0.002). However, only
ERBB2 mRNA expression (as a continuous variable) and number of prior HER2-targeted lines, but
not HER2 IHC expression or hormone-receptor status, were found independently associated with
response (Table 2).
Table 2. Univariable and multivariable logistic regression analyses of overall response.
Univariate
Odds Ratio
(95%CI)
Clinicopathological Variable
Hormone-receptor status
De-novo metastatic disease
Visceral disease
Brain involvement
HER2 IHC
negative
ref
positive
0.37 (0.14–0.95)
no
ref
yes
0.99 (0.40–2.49)
no
ref
yes
0.69 (0.19–2.50)
no
ref
yes
0.98 (0.39–2.49)
≤2+
ref
3+
1.84 (1.26–2.69)
ERBB2 (continuous)
Prior lines HER2-targeted therapy
1.73 (1.25–2.39)
0–1
ref
≥2
0.06 (0.01-0.50)
p
0.038
Multivariable
Odds Ratio
p
(95%CI)
ref
0.152
0.39 (0.11–1.41)
0.990
0.577
0.966
0.002
ref
0.257
1.32 (0.82–2.13)
0.001
0.009
1.95 (1.22–3.12)
ref
0.02 (0.002-0.23)
0.006
0.002
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2.2. Identification of an Optimized ERBB2 mRNA Cutoff
Using T-DM1 response data in advanced HER2+ BC (i.e., PR and CR vs. stable disease [SD] and
progression of disease [PD]), an optimized ERBB2 mRNA cutoff was identified based on Fisher’s exact
test. This cutoff maximized the area under the curve (AUC) of the receiver operating characteristic
(ROC) curve (AUC = 0.701, sensitivity: 100%, specificity: 41.5%) (Figure 2a). The cutoff was ERBB2
log2 value = 1.483 and it classified 60 tumors (77.9%) as ERBB2-high and 17 (22.1%) as ERBB2-low
(Figure 2b). As expected, the ERBB2-high group had a response rate of 60%, whereas the response rate
in the ERBB2-low group was 0%. ERBB2-high, as compared to ERBB2-low, was significantly associated
with higher response rates, both in the HER2 IHC 3+ subgroup (67% vs. 0%, Fisher exact p = 0.017)
and in the HER2 IHC 0–2+ subgroup (42% vs. 0%, Fisher exact p = 0.037).
Finally, the ERBB2-high group had a better PFS compared to the ERBB2-low group (median PFS 6.2
months vs. 2.93 months; hazard ratio = 0.36, 95% CI 0.20–0.65, p = 0.001) (Figure 2c), even after correction
by number of prior lines of HER2-targeted therapy in the metastatic setting (hazard ratio = 0.38, 95%
CI 0.21–0.70, p = 0.002).
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Figure 2. ERBB2 mRNA expression predicts T-DM1 response and survival in metastatic HER2+ BC.
(a) ROC curve to identify ERBB2 mRNA cutoff of response to T-DM1. (b) ERBB2 mRNA levels in
patients with progressive disease (PD) or stable disease (SD) (n = 41) vs. patients achieving complete
response (CR) or partial response (PR) (n = 36). p-value was determined using a two-tailed unpaired
t-test. The ERBB2 mRNA cutoff is shown as a red line. (c) Kaplan–Meier estimate of progression-free
survival (PFS) using the ERBB2 mRNA cutoff.
2.3. Validation of ERBB2 mRNA Expression in Early-Stage HER2+ BC Treated with Neoadjuvant T-DM1
To further validate the ERBB2-based assay as a predictor of response to T-DM1 in the neoadjuvant
setting, we assessed gene expression and pCR data from 158 patients treated with T-DM1 (alone or in
combination with endocrine therapy) in the WSG-ADAPT HER2+/hormone receptor-positive (HR+)
Phase II Trial [13]. Since gene expression in the WSG-ADAPT trial was determined using a different
nCounter CodeSet and house-keeping gene list, we estimated where our ERBB2 cutoff would fall.
To accomplish this, we first determined the percentile of ERBB2 mRNA expression corresponding to our
ERBB2 cutoff (i.e., 1.483) in 77 HER2+/HR+ primary tumors from our previously published PAMELA
trial [14], since this cohort is similar to the WSG-ADAPT cohort. Our ERBB2 cutoff corresponded to
the 50th percentile in primary HER2+/HR+ tumors.
We then applied the 50th percentile of ERBB2 mRNA expression as the cutpoint to define
ERBB2-high from ERBB2-low in the 161 patients of WSG-ADAPT trial. The overall pCR rate was 34.8%
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(56/161). When the ERBB2 cutoff was evaluated, the pCR rate in the ERBB2-high group was 42.9%
(33/77) and in the ERBB2-low group was 27.4% (23/84) (sensitivity = 58.9%; specificity = 58.1%; odds
ratio = 2.0; p-value = 0.041). Altogether, these results confirmed a significant association between
ERBB2 mRNA levels and response to neoadjuvant T-DM1.
2.4. Exploring ERBB2 mRNA Expression and In Vitro Response to T-DM1
Next, we evaluated the expression of ERBB2 and the effects of T-DM1 across 6 HER2+ (HCC1954,
ZR-75-30, BT-474, SK-BR3, HCC1569 and MDA-MB-453) and 3 HER2-negative BC cell lines (MCF7,
T-47D and MDA-MB-468) (Figure 3a,b). ERBB2 mRNA expression varied substantially across cell
lines and similarly to patient’s tumors (interquartile range of 6.16 across all cell lines and 0.60 within
the HER2+ cell lines). As expected, HER2+ cell lines showed higher ERBB2 expression than the
HER2-negative cell lines (mean 1.39 vs. −4.64, p = 0.0002) (Figure S2A). HER2+ cell lines were also
more responsive to T-DM1 than HER2-negative cell lines (Figure 3c and Figure S2B), consistently with
findings reported by others [15,16]. Response was defined as the decrease in cell viability (%) at 72 h
of T-DM1 treatment. HER2+ cell lines showed greater response to T-DM1 than HER2-negative cell
lines (mean response 54.71% vs. 5.24%, p = 0.008) (Figure S2C). Importantly, we observed response to
T-DM1 in all cell lines with ERBB2 mRNA levels above the cutoff. In addition, correlation between
ERBB2 mRNA and T-DM1 response was observed across the 9 cell lines (coefficient = 0.7, p = 0.05)
(Figure 3d). This correlation coefficient suggests that 76.6% of the differences in response across cell
lines may be explained by ERBB2 levels.
2.5. ERBB2 mRNA Expression in BC across the HER2 IHC-Based Groups
To determine the proportion of ERBB2-high tumors across the HER2 IHC groups, we analyzed a
retrospective dataset of 392 tumor samples from HCB with both HER2 IHC status and gene expression
data. As HER2 protein increased, ERBB2 mRNA levels increased as well, and all possible comparisons
(except for 1+ vs. 2+/non-amplified) of ERBB2 expression between groups were statistically significant.
According to our pre-established cutoff, the proportion of ERBB2-high across 0, 1+, 2+/ISH-negative,
2+/ISH-positive and 3+ was 0%, 1.1%, 0%, 9.38% and 76.17%, respectively (Figure 4a).
To provide more evidence of the association of ERBB2 and HER2 IHC expression in BC, we
explored 368 BCs from the TCGA dataset including ERBB2 mRNA expression and HER2 IHC. Since the
methodology to assess ERBB2 mRNA expression in our dataset (nCounter) was different to the TCGA
dataset (RNAseq), the range of ERBB2 mRNA expression was different for each cohort. Therefore, we
estimated where the pre-established cutoff would fall in the TCGA cohort. To do so, we calculated the
median ERBB2 mRNA log2 values for each IHC group in HCB and TCGA cohorts (Figure S3A) and the
Pearson correlation between the two cohorts (Figure S3B). The proportion of ERBB2-high across 0, 1+,
2+/ISH-negative, 2+/ISH-positive and 3+ was 0%, 0.59%, 0%, 25% and 74.04%, respectively (Figure 4b).
The correlation coefficient between these ERBB2-high proportions and the proportions found in our
in-house dataset was 0.975.
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Figure 3. ERBB2 mRNA expression correlates with response to T-DM1 in cell lines. (a) Images of HER2
expression by IHC in 9 BC cell lines (40×). (b) Image of HER2 amplification by ISH (100×). (c) Cell
viability of 9 BC cell lines upon 72 h of treatment with 1.25 µg/mL T-DM1. Data points represent the
mean; error bars represent the standard error of the mean of 3 independent experiments. (d) Spearman
correlation between ERBB2 mRNA expression and response to 1.25 µg/mL T-DM1 expressed as 1-cell
viability (%).
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Figure 4. ERBB2 mRNA expression in BC across the HER2 IHC groups. Distribution of ERBB2 mRNA
levels across HER2 IHC subgroups of the primary BC (a) HCB and (b) TCGA datasets. The proportion
of ERBB2-high tumors is indicated as defined by the ERBB2 mRNA cutoff (shown as a red line). p-values
were determined using one-way analysis of variance.
2.6. ERBB2 mRNA Expression across Cancer Types
In order to determine the proportion of ERBB2-high tumors in other cancer types, we
explored ERBB2 expression for 10,071 tumors of different origins. According to our pre-established
cutoff, ERBB2-high tumors were identified in 15 cancer types including: prostate cancer (0.2%),
lung adenocarcinoma (0.59%), lung squamous cell carcinoma (0.83%), head and neck squamous cell
carcinoma (0.97%), ovarian serous cystadenocarcinoma (1%), colon adenocarcinoma (1.14%), uterine
carcinosarcoma (1.75%), uterine corpus endometrial carcinoma (2.09%), pancreatic adenocarcinoma
(3.39%), rectum adenocarcinoma (3.9%), bladder urothelial carcinoma (3.93%), cervical squamous cell
carcinoma (4.08%), esophageal adenocarcinoma (6.08%), stomach adenocarcinoma (6.32%) and breast
cancer (9.41%) (Figure 5 and Table S1). As expected, we identified a lower proportion of ERBB2-high
tumors in each cancer type as compared to standard IHC/ISH definition of HER2-positivity, thus
potentially selecting tumors sensitive to T-DM1 treatment even in cancer types generally not considered
amenable to treatment with this agent.
Figure 5. ERBB2 mRNA expression across cancer types. Distribution of ERBB2 mRNA levels across
cancer-types TCGA datasets. The ERBB2 mRNA cutoff is shown as a red line. Abbreviations are shown
in Table S2.
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2.7. ERBB2 mRNA Expression in HER2+ Gastric Cancer Treated with T-DM1
We retrospectively studied the case of a 42-year-old male with a gastroesophageal adenocarcinoma
diagnosed at HCB. In September 2011, a total esophagectomy and cervical esophago-gastric anastomosis
was performed. Ascites and pleural effusion positive cytology adenocarcinoma were observed after
surgery, and the patient was diagnosed with a HER2 3+ pT2pN3pM1 stage IV gastroesophageal
junction adenocarcinoma. The patient subsequently received 6 cycles of first-line treatment with
cisplatin plus 5-fluorouracil and trastuzumab achieving a radiological CR.
After 10 months, the patient presented with lung and bone PD and was enrolled in the GATSBY
phase II/III trial [17] and treated with T-DM1 (2.4 mg/kg weekly) monotherapy. Since July 2013, he
received 8 cycles of T-DM1 and obtained a PR (Table 3 and Figure S4) and a time-to-progression of
5.4 months. Of note, median PFS and ORR in GATSBY’s T-DM1 arm was 2.7 months and 20.6%,
respectively [17]. Concordant with the efficacy results obtained in our patient, ERBB2 mRNA levels
measured in the primary tumor were high (i.e., 2.99). Upon PD, the patient received third-line docetaxel
monotherapy achieving SD until July 2014 when he presented with a central nervous system PD.
The patient was lost to follow-up in November 2014.
Table 3. Response evaluation criteria in solid tumors (RECIST) table for a gastric cancer case before
and during T-DM1 treatment.
Target Lesion
Screening
Pre Cycle 3
Pre Cycle 5
Pre Cycle 7
Pre Cycle 9
Right upper lobe lung metastasis
14 mm
8 mm
8 mm
8 mm
10 mm
Left upper
lobe lung metastasis
10 mm
10 mm
10 mm
10 mm
12 mm
Mesentheric adenopathy
17 mm
11 mm
11 mm
11 mm
18 mm
Retroperitoneal adenopathy
18 mm
9 mm
9 mm
9 mm
17 mm
Total
59 mm
38 mm
38 mm
38 mm
57 mm
NA
39% reduction
39% reduction
39% reduction
36% increase
NA
PR
Maintained PR
Maintained PR
PD
Response
3. Discussion
As an increasing number of HER2-targeted agents are becoming available in clinical practice,
biomarkers are increasingly needed that can predict the response to specific anti-HER2 agents beyond
the classic IHC/ISH definition of HER2-positivity. In this context, HER2 3+ tumors have been reported
to benefit more from T-DM1 than other IHC groups in retrospective and prospective studies [7,18].
Moreover, benefit associated with the use of post-neoadjuvant T-DM1 as compared to trastuzumab in
the KATHERINE trial appeared to more marked in HER2 3+ than in HER2 2+ tumors [19]. Greater
benefit to T-DM1 in HER2 3+ tumors has also been reported in the KATE2 trial [20] and other cancer
types [21,22].
ERBB2 mRNA expression has been previously associated with a more pronounced T-DM1 benefit
in several randomized clinical trials which tested the use of T-DM1 as compared to other HER2-targeted
treatments in HER2+ metastatic BC [23–25]. In the randomized EMILIA trial, which compared T-DM1
and capecitabine-lapatinib (CL) for pretreated metastatic HER2+ BC patients, patients with tumor
ERBB2 mRNA levels above median showed a greater benefit from the use of T-DM1 in terms of ORR
and overall survival (OS). However, T-DM1 treatment, compared with CL, reduced the risk of PD to a
similar degree regardless of tumor ERBB2 mRNA levels and tests for interaction between treatment
and ERBB2 mRNA levels were not statistically significant (p = 0.07). However, tests were exploratory
and not powered to detect an interaction [23].
Moreover, data from the randomized TH3RESA trial, which compared T-DM1 vs. treatment of
physician choice for pretreated metastatic HER2+ BC patients, confirmed that patients with higher
ERBB2 mRNA levels benefited more from T-DM1 than patients with lower ERBB2 mRNA levels [24].
Similar results confirming an association between higher ERBB2 mRNA levels and increased T-DM1
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benefit, both in terms of ORR and PFS, were also reported for several randomized phase II trials [25–27].
Furthermore, the impact of ERBB2 mRNA levels on T-DM1 benefit has been reported in other cancers
beyond BC. A translational study [21] evaluating gastric cancer samples of the GATSBY trial [17]
demonstrated more benefit to T-DM1 in terms of PFS in patients with tumors with higher ERBB2
mRNA levels [21].
In addition, recently presented biomarker data from the post-neoadjuvant KATHERINE trial have
been reported showing that patients with high ERBB2 mRNA levels (above median) at surgery have a
worse outcome than patients with low ERBB2 mRNA levels when treated with adjuvant trastuzumab,
but not when treated with adjuvant T-DM1. In fact, while both patients with ERBB2 mRNA levels above
and below median levels benefited from switching to T-DM1, those with higher ERBB2 mRNA levels
(above median) benefited more from T-DM1 than those with lower ERBB2 mRNA levels, potentially
questioning the use of median mRNA expression value as cutoff in this setting [28].
Our results not only confirm the association between ERBB2 mRNA expression and T-DM1
benefit in a more heterogeneous real-world setting, but also highlight the relevance of a quantitative
method as a better method to predict response to T-DM1 by proposing a cutoff for selecting patients
responsive to T-DM1 both in the metastatic and neoadjuvant settings. Other HER2-targeted ADCs
are entering clinical practice, for instance DS-8201, which has shown activity in HER2-low advanced
BC [12,29]. Therefore, we might expect that different ERBB2 cutoffs will be needed for different ADCs.
In this context, the use of a quantitative method such as ERBB2 mRNA expression, which offers
the opportunity to identify different cutoffs, might potentially improve treatment personalization.
Moreover, a quantitative method as ERBB2 mRNA expression might recapitulate tumor heterogeneity
in a single, easily manageable assay.
Our study has several limitations. First, the study cohort is retrospective and only involved
a limited number of patients who were treated according to everyday clinical practice, thus being
heterogeneous, both in previous lines of treatment received and in the kind of histological samples
available (primary tumor vs. metastasis). Despite this, our analysis was able to clearly identify ERBB2
mRNA expression as the main predictor of responsiveness together with number of previous lines of
HER2-targeted treatment. Moreover, we validated ERBB2 mRNA as a predictor of response in the
neoadjuvant setting. This highlights the clinical importance of ERBB2 mRNA expression. Second, we
could not address if the biomarker works better when primary or metastatic tumor samples are used.
Third, our data in non-BC-types is currently in the hypothesis-generating stage.
4. Materials and Methods
4.1. Patient Datasets and Tumor Samples
This study analyzed a retrospective cohort of 77 HER2+ (as defined by standard guidelines [4])
advanced/metastatic BC patients treated with T-DM1 between January 2013 and November 2019 in two
independent institutions: Hospital Clínic of Barcelona (HCB) (n = 26) and Istituto Oncologico Veneto
(IOV) in Padova (n = 51). One formalin-fixed paraffin-embedded (FFPE) tumor sample per patient
was selected: if available, a biopsy of metastatic site nearest in time to start of T-DM1 was preferred
(n = 38); otherwise primary tumor sample was used (n = 39), favouring pre-treatment biopsy over
surgical sample for patients treated with neoadjuvant therapy. Gene expression was also assessed in
77 primary HER2+/HR+ BC of the PAMELA trial [14], and gene expression and pathological complete
response (pCR) data was evaluated in 161 HER2+ primary samples of the T-DM1 arms (A&B) of the
WSG-ADAPT HER2+/HR+ Phase II Trial [13]. In addition, we evaluated 392 primary BCs from HCB
with available HER2 IHC status and gene expression data evaluated at the nCounter platform; 368
primary BCs from The Cancer Genome Atlas (TCGA) with HER2 IHC status and ERBB2 RNASeqv2
data; and 10,071 TCGA pan-cancers with ERBB2 RNASeqv2 data. Finally, we analyzed a primary
tumor sample of a patient with advanced gastric cancer treated with T-DM1 in the GATSBY trial [17]
at HCB.
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4.2. In Vitro Cell Lines and T-DM1
The BC cell lines BT-474, HCC1569, HCC1954, MCF7, MDA-MB-453, MDA-MB-468, SK-BR3,
T-47D and ZR-75-30 were purchased from the American Type Culture Collection. All cell lines were
maintained as recommended by the suppliers. T-DM1 was provided as remnant of the product used in
common clinical practice by the oncology pharmacy Service at HCB.
4.3. HER2 Immunohistochemistry and Fluorescent In Situ Hybridization
HER2 status was re-assessed in 74 of 77 FFPE tumors of the T-DM1 HCB/IOV cohort and FFPE BC
cell line pellets by either IHC and/or in situ hybridization (ISH) according to the American Society of
Clinical Oncologists (ASCO)/College of American Pathologists (CAP) guidelines. IHC was performed
on 2-µm-thick sections using anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody kit (Ventana
Medical Systems Inc., Oro Valley, AZ, USA) and ISH for HER2 was performed on 4-µm-thick sections
using the FDA-approved XL ERBB2 (HER2/NEU) AMP (MetaSystems Probes, Altlußheim, Germany)
according to manufacturer’s instructions.
4.4. In Vitro Cell Viability Assay
BC cell lines were plated in triplicate at 4000 cells/well in 96-well plates. Cells were then treated
with 1.25 µg/mL T-DM1. Cell viability was determined 72 h after treatment using CellTiter 96 AQueous
One Solution Cell Proliferation Assay (MTS) (Promega Corporation, Madison, Wisconsin, USA)
following the manufacturer’s instructions, and quantified using the Gen5 Microplate Reader and
Imager Software (BioTek, Winooski, VT, USA). Data were analyzed using GraphPad Prism 5 software
(GraphPad, San Diego, CA, USA).
4.5. RNA Extraction
RNA samples were extracted from biopsy and surgical tumor FFPE material using the High
Pure FFPET RNA isolation kit (Roche) following manufacturer’s protocol. FFPE slides with at least
10% tumor cells and 4 mm2 of tissue were used for each tumor specimen, and macrodissection was
performed to avoid contamination with normal breast tissue if needed. Cell line RNA samples were
extracted using the RNeasy Mini Kit (Qiagen, Hilden, Alemanya). RNA samples were quantified at
the NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
4.6. Gene Expression Analysis
The nCounter platform (NanoString Technologies, Seattle, WA, USA) was used to analyze RNA
samples from tumors and cell lines. A minimum of 100 ng of total RNA was used to measure the
expression of 50 genes of the PAM50 intrinsic subtype predictor assay and 5 housekeeping genes
(ACTB, MRPL19, PSMC4, RPLP0, and SF3A1). Expression counts were then normalized using the
nSolver 4.0 software (nanoString, Seattle, WA, USA) and custom scripts in R 3.4.3 (R Foundation,
Vienna, Austria) [30].
4.7. Statistical Analysis
Univariate and multivariable logistic regression analyses were used to investigate the association
of each variable with overall response. Odds ratios and 95% confidence intervals (CIs) were
calculated for each variable. An optimized cutoff of gene expression was identified to predict
overall response. Univariate and multivariable Cox proportional hazard regression analyses were
performed to investigate the association of each variable with PFS. All statistical analyses were carried
out using the R software version 3.4.3.
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5. Conclusions
To conclude, our study presents a clinically applicable assay to help identify patients most likely
to benefit from T-DM1, regardless of HER2 status. In addition, the assay could help identify patients
most likely to benefit from other HER2-targeted ADCs across cancer types.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/7/1902/s1,
Table S1: Proportions of ERBB2-high tumors across cancer types, Table S2: Table of abbreviations, Figure S1:
Distribution of ERBB2 mRNA levels across HER2 IHC subgroups of tumors of the T-DM1 cohort, Figure S2:
ERBB2 mRNA levels and T-DM1 response in BC cell lines, Figure S3: Determination of an ERBB2 mRNA cutoff for
the TCGA dataset, Figure S4: Partial response to T-DM1 in gastric cancer patient.
Author Contributions: Experimental study design: G.G., F.B.-M. and A.P. Data acquisition and analysis:
G.G., F.B.-M., B.G.-F., T.P., N.C., T.S., R.K., O.G., D.M, V.T., D.P., P.G., L.B., M.C. (Miriam Cuatrecasas), M.C.
(Mathias Christgen), H.K., T.G. Data interpretation: G.G., F.B.-M., B.G.-F., T.P., N.C., T.S., R.K., O.G., D.M., L.P., V.T.,
D.P., M.V.D., B.A., M.M., P.G., L.B., M.C. (Miriam Cuatrecasas), M.C. (Mathias Christgen), H.K., I.M.-E., P.V., D.S.,
T.G., M.V.D., P.C., N.H., V.G., A.P.; Writing of the manuscript: G.G., F.B-M. and A.P. Review of the manuscript: all
authors. All authors have read and agreed to the published version of the manuscript.
Funding: This study has received funding from Instituto de Salud Carlos III—PI16/00904 and PI19/01846 (to A.P.),
Breast Cancer Now—2018NOVPCC1294 (to A.P.), Breast Cancer Research Foundation-AACR Career Development
Awards for Translational Breast Cancer Research 19-20-26-PRAT (to A.P.), Fundació La Marató TV3 201935-30
(to A.P.), the European Union’s Horizon 2020 research and innovation programme H2020-SC1-BHC-2018-2020
(to A.P.), Pas a Pas (to A.P.), Save the Mama (to A.P.), Fundación Científica Asociación Española Contra el
Cáncer AECC_Postdoctoral17-1062 (to F. B-M), Generalitat de Catalunya Peris PhD4MD 2019 SLT008/18/00122
(to N.C.), DiSCOG—University of Padova DOR 1721185/17 and DOR 1830512/18 (to MV.D.), Conquer Cancer
Foundation/Shanken Family Foundation -YIA in Breast Cancer 2019 (to G.G.).
Conflicts of Interest: Potential conflicts of interest are the following: A.P. reports consulting fees from Nanostring
Technologies Roche, Pfizer, Novartis and Daiichi Sankyo outside the submitted work. A.P. is listed as an inventor
on a patent application on HER2 as a predictor of response to dual HER2 blockade in the absence of cytotoxic
therapy (WO2018/103834A1). MV.D. reports lecture fees and honoraria for participation on advisory boards
from Roche, Genomic Health, Eli Lilly, and Celgene outside the submitted work. PF.C. reports honoraria for
participation on advisory boards from Eli Lilly, Novartis, AstraZeneca, Tesaro, Roche Genentech, Daiichi Sankyo,
and BMS, and research grants to the Institution from Novartis, Roche Genentech, Merck KGaA, and BMS outside
the submitted work. V.G. reports lecture fees and honoraria for participation on advisory boards from Eli Lilly,
Roche Genentech and Novartis, honoraria for participation on Speakers bureau from Eli Lilly and Novartis outside
the submitted work. N.H. reports fees for consulting and/or lectures Novartis and Roche outside the submitted
work and minority ownership in WSG (Westdeutsche Studiengruppe).
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