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Breast Cancer Research
NSABP FB-10: aphase Ib/II trial evaluating
ado-trastuzumab emtansine (T-DM1)
withneratinib inwomen withmetastatic
HER2-positive breast cancer
Samuel A. Jacobs1*, Ying Wang1, Jame Abraham1,2, Huichen Feng1, Alberto J. Montero1,2,3, Corey Lipchik1,
Melanie Finnigan1, Rachel C. Jankowitz1,4,5, Mohamad A. Salkeni1,6,7, Sai K. Maley1, Shannon L. Puhalla1,8,9,
Fanny Piette10, Katie Quinn11, Kyle Chang11, Rebecca J. Nagy11, Carmen J. Allegra1,12, Kelly Vehec1,
Norman Wolmark1,8, Peter C. Lucas1,8,9,13, Ashok Srinivasan1,14 and Katherine L. Pogue‑Geile1
Abstract
Background We previously reported our phase Ib trial, testing the safety, tolerability, and efficacy of T‑DM1 + ner‑
atinib in HER2‑positive metastatic breast cancer patients. Patients with ERBB2 amplification in ctDNA had deeper
and more durable responses. This study extends these observations with in‑depth analysis of molecular markers
and mechanisms of resistance in additional patients.
Methods Forty‑nine HER2‑positive patients (determined locally) who progressed on‑treatment with trastu‑
zumab + pertuzumab were enrolled in this phase Ib/II study. Mutations and HER2 amplifications were assessed
in ctDNA before (C1D1) and on‑treatment (C2D1) with the Guardant360 assay. Archived tissue (TP0) and study
entry biopsies (TP1) were assayed for whole transcriptome, HER2 copy number, and mutations, with Ampli‑Seq,
and centrally for HER2 with CLIA assays. Patient responses were assessed with RECIST v1.1, and Molecular Response
with the Guardant360 Response algorithm.
Results The ORR in phase II was 7/22 (32%), which included all patients who had at least one dose of study therapy.
In phase I, the ORR was 12/19 (63%), which included only patients who were considered evaluable, having received
their first scan at 6 weeks. Central confirmation of HER2‑positivity was found in 83% (30/36) of the TP0 samples.
HER2‑amplified ctDNA was found at C1D1 in 48% (20/42) of samples. Patients with ctHER2‑amp versus non‑amplified
HER2 ctDNA determined in C1D1 ctDNA had a longer median progression‑free survival (PFS): 480 days versus 60 days
(P = 0.015). Molecular Response scores were significantly associated with both PFS (HR 0.28, 95% CI 0.09–0.90,
P = 0.033) and best response (P = 0.037). All five of the patients with ctHER2‑amp at C1D1 who had undetectable
ctDNA after study therapy had an objective response. Patients whose ctHER2‑amp decreased on‑treatment had bet‑
ter outcomes than patients whose ctHER2‑amp remained unchanged. HER2 RNA levels show a correlation to HER2
CLIA IHC status and were significantly higher in patients with clinically documented responses compared to patients
with progressive disease (P = 0.03).
*Correspondence:
Samuel A. Jacobs
jacobssa1945@gmail.com
Full list of author information is available at the end of the article
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Page 2 of 13
Jacobsetal. Breast Cancer Research (2024) 26:69
Conclusions The following biomarkers were associated with better outcomes for patients treated with T‑DM1 + ner‑
atinib: (1) ctHER2‑amp (C1D1) or in TP1; (2) Molecular Response scores; (3) loss of detectable ctDNA; (4) RNA levels
of HER2; and (5) on‑treatment loss of detectable ctHER2‑amp. HER2 transcriptional and IHC/FISH status identify HER2‑
low cases (IHC 1+ or IHC 2+ and FISH negative) in these heavily anti‑HER2 treated patients. Due to the small number
of patients and samples in this study, the associations we have shown are for hypothesis generation only and remain
to be validated in future studies.
Clinical Trials registration NCT02236000
Keywords Metastatic breast cancer, ctDNA HER2 amplification, Clinical trial, Neratinib + t‑DM1
Introduction
In 2013, T-DM1 was the first HER2-targeted antibody–
drug conjugate (ADC) granted FDA-approval for late-
stage metastatic breast cancer after prior trastuzumab. In
2019, T-DM1 was approved as post-neoadjuvant therapy
in patients with residual disease based on the KATHER-
INE trial, demonstrating that post-neoadjuvant T-DM1
was statistically more beneficial than trastuzumab,
preventing recurrence of invasive disease or deaths in
patients with residual disease in breast or lymph nodes
after treatment with trastuzumab ± pertuzumab (haz-
ard ratio for invasive disease or death, 0.05: 95% CI
0.039–0.64; P < 0.001) [1]. KATHERINE required archi-
val HER2-positivity but did not mandate HER2 status
at study entry. Because multiple studies have confirmed
that HER2 status is plastic with conversion of HER2-pos-
itive disease to HER2-low (IHC = 0–1+ or IHC 2+ /FISH-
negative) under pressure of therapy [2–4], this raised the
question of ADC efficacy in HER2-low patients—either
de novo (HR + /HER2-negative) or acquired from con-
version of HER2-amplified to HER2-low. ere are now
several breast cancer-targeting ADCs in the pipeline in
addition to the newly approved trastuzumab deruxtecan
(T-DXd). Initial approval of T-DXd was for metastatic
HER2-positive breast cancer after prior progression on
multiple lines of anti-HER2 therapy (DESTINY-Breast01)
[5]. In DESTINY-Breast03, T-DXd improved PFS and
OS compared to T-DM1 [6] in patients with metastatic
disease with progression on trastuzumab. In heavily pre-
treated HER2-low breast cancer patients, T-DXd was
evaluated in a single arm phase II study. e objective
response rate (ORR) to T-DXd by central review was
37%, with median duration of response of 10.4months
[7]. DESTINY-Breast04, a randomized, multicenter trial
in patients with unresectable or metastatic HER2-low
breast cancer, reported highly significant improvements
in PFS and OS in patients receiving T-DXd compared to
physician choice of treatment [8]—a particularly striking
observation, because neither trastuzumab nor T-DM1
has shown consistent activity in HER2-low popula-
tions [9]. HER2 status (expression, mutation, amplifica-
tion) is thus emerging as a predictor of clinical efficacy
for anti-HER2-therapy. In our NSABP phase Ib trial of
HER2-targeted therapies using T-DM1 + neratinib in
HER2-positive patients, we showed a discordance in
HER2 status between archival tissue and a liquid biopsy
obtained at study entry. Loss of ctHER2-amp occurred
in 63% (17 of 27) patients. Deeper and more durable
responses were observed with T-DM1 + neratinib in
patients with ctHER2-amplification [10]. We now report
on an expanded cohort. Our aims were to: (1) confirm
activity of T-DM1 + neratinib in patients progressing on
a taxane with trastuzumab + pertuzumab (HP), (2) evalu-
ate discordance in HER2 amplification between archived
tissue, contemporaneous tissue, and blood, and (3) com-
pare mutation and gene-expression profiles at different
time points. Finally, in a subset of patients, we assessed
response by RECIST 1.1 with the Guardant Molecular
Response score [11, 12].
Methods
Trial design
FB-10 was a single-arm, nonrandomized, unblinded
clinical trial approved by participating institutions’ insti-
tutional review boards. Written informed consent was
required. FB-10 was conducted according to Good Clini-
cal Practices and the Declaration of Helsinki.
e phase Ib trial was a dose escalation study evaluat-
ing T-DM1 + neratinib in women with metastatic HER2-
postive breast cancer based on local determination of
HER2. Patients received 3.6mg/kg T-DM1 intravenously
on a 3-week cycle and oral neratinib was taken daily in
one of four dose cohorts (120, 160, 200 and 240 mg).
Twenty-seven patients enrolled, with 5 experiencing a
dose-limited toxicity. ree withdrew early for other
reasons (Fig. 1). Nineteen patients were evaluable for
response, which required follow-up imaging after the
second cycle of treatment (6weeks). e recommended
phase II dose of neratinib was determined to be 160mg/d
[10]; however, we did not detect a dose response, i.e., ner-
atinib at 120mg/d was as effective as higher doses and
less toxic. [10]
e phase II expansion included all patients (N = 22)
who received at least one dose of study therapy in
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Jacobsetal. Breast Cancer Research (2024) 26:69
the analysis of safety and efficacy. Eligibility criteria
were identical in phase Ib and phase II [10]. All eligi-
ble patients had prior HP and a taxane as neoadjuvant
therapy or for de novo metastatic disease, had measur-
able disease, were ECOG PS ≤ 2, with adequate hema-
tologic, renal, and liver function. Patients with known
stable brain metastases were eligible. Brain imaging at
entry was not required. Treatment in phase II included
T-DM1 at 3.6 mg/kg iv q 3 weeks and neratinib at
160 mg/day. Primary diarrhea prophylaxis was man-
dated as described in phase I [10].
Safety assessment
Safety assessment was similar in phase Ib and phase II
including physical examination, interim history, and
laboratory assessments. Patients remained on treat-
ment until progressive disease or discontinuation
because of withdrawal, physician discretion or toxicity.
For phase Ib patients, adverse event (AE) assessment
occurred on days 1, 8, and 15 of cycle 1; on day 1 of
each cycle and for 30days after therapy discontinuation
or when alternate therapy began. Phase II AE assess-
ments were made on day 1 of each cycle.
Response evaluation
In phase Ib, patients were assessed for best response
beginning with their first follow-up scan after 2 cycles
(6 weeks). Response by RECIST v1.1 was complete
response (CR), partial response (PR), stable disease
(SD), or progression (PD). In phase II, imaging stud-
ies were performed after every 3 cycles (9weeks). e
clinical benefit rate included all CR, PR, and SD patients
with duration ≥ 180 days. Patients with stable disease
of < 180 days were included with progressive disease
patients. A confirmatory scan at least one month after
the best response was not required in this study, perhaps
accounting for partial responses of short duration in sev-
eral patients.
Blood andtissue collection
Blood samples were required before treatment at cycle 1,
day 1 (C1D1), and after treatment at cycle 2day 1 (C2D1)
for all patients in phase Ib and II (Fig.1A). Archived tis-
sue (TP0) of diagnostic blocks or slides were required on
all phase Ib and II patients which included 27 patients
from phase Ib and 22 patients in phase II. (Fig.1B). Con-
temporaneous biopsy specimens or slides at study entry
(TP1) were optional in phase Ib but in phase II, after
Fig. 1 Remark Diagram of Blood and Tissue Samples: NSABP FB‑10. A Blood samples collected from patients enrolled into FB‑10 phase Ib
and phase II, and successful assays for ctDNA analysis of HER2 amplification with Guardant360 assays. B Tissue samples collected from patients
enrolled into FB‑10 and their samples that were profiled for mutations and whole transcriptomic analysis and for ERBB2 amplification status
with CLIA and AmpliSeq assays. C The timing and type of sample collections (tissue: TP0 or TP1, or blood: C1D1 or C2D1) are shown
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Jacobsetal. Breast Cancer Research (2024) 26:69
enrollment of the first 6 patients, the study was amended
to require a study entry biopsy. e timing and type of
collections of samples are shown in Fig.1C.
ctDNA assessment
Samples were analyzed by the Guardant360 assay
(Fig.1A), which detects single-nucleotide variants, indels,
fusions, and copy number alterations in 74 genes. For
HER2 amplification, a cutoff of ≥ 2.14 was used. Where
amplification could not be determined because of failed
assays or no blood, samples were categorized as indeter-
minant. Guardant Health (Guardant360 assay) is Clinical
Laboratory Improvement Amendments (CLIA)-certified,
College of American Pathologists-accredited, New York
State Department of Health-approved laboratory.
ctDNA molecular response
Guardant360 Molecular Response is a next generation
sequencing (NGS)‐based liquid biopsy that assesses
changes in tumor‐derived cell‐free DNA (ctDNA)
between baseline and an early on‐treatment timepoint
in patients with solid tumor malignancies. It employs
an algorithm to identify informative somatic single
nucleotide variants (SNVs), insertions/deletions and
gene fusions and calculates the percent ctDNA change
between the two timepoints based on the mean vari-
ant allele frequency (VAF) between two samples (mean
VAF2/mean VAF1) -1 × 100%. Using the Molecular
Response panel and the Guardant bioinformatics pipe-
line, the change in ctDNA levels between baseline and
the initial follow-up scan (6weeks in phase I and 9weeks
in phase II) was calculated and the change in VAF deter-
mined. Molecular Response is calculated as the ratio of
mean VAF on-treatment to baseline with a cutoff of 50%.
Decreases in ctDNA of 50%‐100% during this timeframe
are associated with clinical benefit in patients on anti‐
cancer therapies [11, 13, 14]. Kaplan–Meier curves for
PFS are generated for patients above and below a Molec-
ular Response cut off. [11]
Isolation ofnucleic acid
Tumor regions, defined by a certified pathologist, were
macrodissected. DNA and RNA were isolated using the
Qiagen AllPrep DNA/RNA kit, following the manufac-
turer’s recommendations but eliminating the xylene wash
in the first step. Separate tissue sections were used for
RNA and DNA isolation.
Whole transcriptomic proling
10–30ng of RNA from the phase II samples was reverse
transcribed. cDNA libraries were constructed using
whole transcriptomic Ampli-Seq kits, following the
manufacturer’s instructions without a fragmentation step
due to the small size of the RNAs. is same method did
not work well for the phase Ib RNAs. To overcome this
problem, phase Ib RNAs were made library-ready via the
HTG EdgeSeq system and the HTP panel, which includes
probes to interrogate 19,398 genes representing most of
the human transcriptome (details in Additional file1).
Breast cancer molecular subtypes were determined
by applying the AIMs signature [15]. e 8-gene trastu-
zumab-benefit groups were determined using a validated
signature. [16, 17]
HER2 amplication status andanalysis ofvariants intissues
DNA sequencing was performed using a custom Ampli-
Seq panel referred to as the NAR panel, amplifying 3,847
amplicons with 94.25% coverage of exons from 117 genes
in HER2-activated pathways (Additional file1: TableS1).
e panel was designed using the ermo Fisher Ion
AmpliSeq™ Designer tool (https:// www. ampli seq. com).
Libraries were constructed using 10ng of DNA using the
Ion AmpliSeq™ kit for Chef DL8. e Ion Chef instru-
ment was used to template and load samples on Ion 550
chips. Up to 32 samples were barcoded, pooled, and
sequenced on the S5 sequencer (ermoFisher) following
manufacturer’s instructions.
We have used two different criteria to identify vari-
ants in FB-10 tissue. For a conservative approach to vari-
ant selection, we selected variants with VAF ≥ 10% and
for a less restrictive option we selected variants with
VAF ≥ 5%. Additional details are included in Additional
file1 and the rationale for these approaches is discussed.
Ion Torrent data and the Ion Reporter software were
used to determine HER2 copy number.
HER2 IHC FISH
HER2 status was also assessed in tissue samples with IHC
and reflexively for FISH at the discretion of the Director
at the CLIA laboratory (Magee Women’s Hospital, Uni-
versity of Pittsburgh Medical Center). Nine samples were
equivocal IHC 0 or 1+ due to poor tissue quality prompt-
ing examination with FISH. Based on FISH, four were
included as HER2-positive.
Statistical analysis
Phase Ib safety, tolerability, efficacy, and recommended
phase 2 dose (RP2D) of neratinib in combination with
T-DM1 was previously reported [10]. In the phase II
expansion cohort, in which neratinib was administered
at the RP2D of 160mg/d, the intention was to confirm
clinical efficacy and tolerability of the combination and
to extend the correlative findings. Given the small sample
size, the endpoint analyses remain descriptive.
e aim of the single-arm phase II expansion was to
rule out the null hypothesis that the ORR was 25% with
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Jacobsetal. Breast Cancer Research (2024) 26:69
the alternative hypothesis of an ORR of 45%. With these
assumptions, the sample size required was 22 and the
decision rules are to declare success if > 8 responses; to
declare failure if < 7 responses; and to consider the trial
inconclusive if 7 or 8 responses (7/22 = 32%, 8/22 = 36%).
At the outset of the study, we did not anticipate the large
number of patients with loss of HER2-amplification in
blood as determined by the Guardant assay. us, the
subset analyses based upon HER-amplification detected
in blood were performed post-hoc.
Results
Patient characteristics
In the phase Ib portion of this study, 27 patients were
enrolled between February 2015 and July 2017. Nineteen
patients were evaluable having had at least one follow-up
imaging study; three patients withdrew from the study
in cycle 1 and 5 patients with a dose-limited toxicity in
cycle 1 did not have an imaging assessment. All phase
II patients who received at least one dose of study drugs
were included in the analysis. Twenty-two patients were
evaluable for toxicity and 20 were evaluated for efficacy
with at least one scan performed after their third cycle.
Two non-evaluable patients who withdrew from the
study did not have their first scan but are included as PD.
Median age was 55.5years (range 32–70). Hormone sta-
tus (ER and/or PR) was positive in 13 patients and nega-
tive in 9. All patients were HER2-positive at baseline by
local determination (Additional file1: TableS3).
Safety assessment
Similar to phase Ib patients, diarrhea was the most fre-
quent toxicity in phase II: grade 2, 6 patients (27%);
grade 3, 8 (36%). Other grade 3/4 toxicities included:
thrombocytopenia, 2 patients (10%); transaminase eleva-
tion, 3 patients (15%); and pneumonitis, 1 patient (5%).
ere were no unanticipated toxicities in the phase II
expansion.
Ecacy
Among 19 evaluable patients in phase Ib, there were 3
CRs and 9 PRs for an ORR of 63% (12/19) [10]. In phase
II, including all patients who received at least one dose
of therapy, there were 2 CRs, 5 PRs for an ORR of 32%
(7/22), and 3 SDs of 180days or longer making the clini-
cal benefit rate (CBR) 45% (10/22). In phase Ib and II,
nine patients had sustained objective responses lasting
approximately 1year or longer (range 343–1453 + days,
Additional file1: TableS4; Additional file2: Table S5).
Treatment was discontinued at or before the first scan in
15 patients for a variety of reasons, including 5 DLTs (all
in phase I) and 10 with clinical progression in phase I and
II.
ctDNA clearance andtreatment response
Because clearance of ctDNA has been associated with
treatment response, we compared the outcomes of
patients who were positive or negative for ctDNA after
study treatment. e response rate among the ctDNA-
positive patients who were still ctDNA-positive after
study therapy was 9/19 (47%), but the ctHER2 DNA-pos-
itive patients who became ctDNA-undetectable at C2D1,
the response rate was 6/6 (100%), demonstrating that the
loss of ctDNA was associated with a very good response.
HER2 amplication intissues andblood
We assessed the HER2 amplification status of TP0 and
TP1 with a CLIA HER2 IHC/FISH assay, and with an
Ampli-Seq NGS assay. ese tissue samples were also
compared to the HER2 amplification status in blood sam-
ples collected at C1D1 and C2D1 (Fig. 2B). ere was
good concordance between the CLIA HER2/FISH and
Ampli-Seq assays (100% in TP1 tissues and 85% in all tis-
sues), which demonstrated the technical accuracy of the
Ampli-Seq. Concordance between TP0 tissue with IHC/
FISH and C1D1 ctDNA was 71% (20/28). Concordance
between C1D1 and C2D1 was 54% (14/26). Anti-HER2
treatment is potentially the causal reason for the discord-
ance between C1D1 and C2D1, which showed a total loss
of ctDNA in some samples and a loss of HER2 amplifica-
tion in others.
Using the Guardant360 assay cut point of 2.14 for
amplification among 43 C1D1 samples (22 in phase
Ib, 21 in phase II), 6 patient samples were indetermi-
nate (including 4 for which somatic mutations were not
detected) (Fig.2B, dark green), 1 was not evaluable (NE),
and 1 other failed quality control. Among the remaining
37 samples, there were 21/37 (57%) patients with ampli-
fication and 17/37 (46%) without. e objective response
(CR, PR) rate was 55% (11/20) in amplified patients
and 41% (7/17) in non-amplified patients. e CBR in
patients with ctHER2-amplification was 12/21 (57%)
and in non-amplified patients it was 8/17 (47%). ere
was one patient with SD who was ctHER2-amp indeter-
minate. Mean duration of response (CBR) was substan-
tially longer in amplified patients, 457days compared to
131days in non-amplified patients (P = 0.008) (Fig. 2A;
Additional file1: TableS4).
We compared progression-free survival (PFS) of
patients whose ctDNA or tumor tissues had HER2 ampli-
fication to patients with no HER2 amplification. Patients
with ctHER2-amp at C1D1 or in their TP1 tumor tis-
sue had a significantly longer PFS than patients with no
HER2 amplification (Fig.3A–E).
In phase I and II there were 26 C1D1 and C2D1 pairs,
15 with and 11 without ctHER2-amp at C1D1. Among
the 15 with ctHER2-amp at C1D1, 14 showed HER2 loss
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Jacobsetal. Breast Cancer Research (2024) 26:69
Fig. 2 Amplification Status of Tissues and Blood: NSABP FB‑10. A Response rates (CR/PR, CBR, and SD) for FB‑10 HER2‑amplified and non‑amplified
patients based on ctDNA results. B HER2‑amplification status of FB‑10 tumor tissues based on CLIA tests (IHC/FISH) and Ampli‑Seq (Tissue)
in baseline (TP0) and study entry ( TP1) samples are shown. HER2‑amplification status was determined in ctDNA at C1D1 and at C2D1
with the Guardant360 assays. Responses, amplification status, and changes in copy number in ctDNA between C1D1 and C2D1 are indicated
as shown in the legend
Fig. 3 Association of HER2‑amplification Status with Patient Outcomes: NSABP FB‑10. A Kaplan‑Meier plots of patients with or without HER2
amplification in ctDNA or in TP0 tissue (B & D), or in TP1 tissue (C & E) based on IHC/FISH (B & C) and on Ampli‑Seq (D & E)
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Jacobsetal. Breast Cancer Research (2024) 26:69
at C2D1 as defined by a loss ≥ 28% of HER2 copy number
or no ctDNA detected. e ORR among these 14 patients
was 71% (10/14). Of the 10 responders, 5 cleared ctDNA
completely by C2D1, 3 had detectable ctDNA but no
ctHER2-amp, and 2 were HER2-positive but the amplifi-
cation level in C2D1had decreased dramatically (Fig.2B;
Additional file2: TableS5). e 2 remaining patients with
detectable ctHER2-amp with no loss of HER2 amplifica-
tion had PD, suggesting that a loss of ctDNA and/or a loss
of HER2 ctDNA amplification was a marker for a good
response to study therapy. However, in 11 patients with
no HER2 ctDNA amplification at C1D1, the ORR was
45% (5/11), indicating that some non-amplified tumors
were responsive to study treatment.
Molecular response byctDNA
We assessed the association between molecular response
and objective radiologic response (Fig.4A). A total of 21
patients (9 phase Ib and 12 phase II) had paired samples
that met criteria for assessment of molecular response.
Criteria included ≥ 1 alteration present in one of the
paired samples plus a mutant molecule count of ≥ 15 in
either sample. Molecular responders demonstrated a
longer PFS compared to non-responders (median PFS 7.4
vs. 2.8, HR 0.28, 95%CI 0.09-0.90, P=0.033 using Wilcox
test). We also examined the association between molecu-
lar response and best RECIST response. Patients with
CR/PR/SD had significantly lower Molecular Response
values compared to patients with PD (P = 0.037; Fig.4B).
Mutations/variants intissues andctDNA
Because ERBB2 is the target of the study therapies, we
have examined both tissue and ctDNA for mutations in
the ERBB2 gene. No ERBB2 variants in tissue at a VAF
of ≥ 10% were observed, however, in ctDNA 3 nonsynon-
ymous, ERBB2 variants (I767M, V777L, and S310Y) were
detected in the C1D1 samples from 3 patients. ese
variants have been associated with sensitivity to neratinib
in breast cancer patients [18]. In this study, the patients
whose tumors had a V777L or a S310Y mutation had a
PR, but the one patient with a I767M mutation had PD
with brain metastasis. e tumor with the I767M muta-
tion also had a P1233L mutation [19]. Interestingly, in
an exhaustive meta-analysis of 37,218 patients, includ-
ing 11,906 primary tumor samples, 5,541 extracerebral
metastasis samples, and with 1485 brain metastasis sam-
ples found that a nearby ERBB2 mutation (P1227S) was
the only mutation restricted to brain metastasis. It is
unknown whether any of these mutations played a role in
the patient responses or the course of disease, but it is of
interest to note them [20].
We examined DNA variants in all available TP0 and
TP1 tissues using our NAR Ampli-Seq panel, which
included ESR1, HER2, and 115 other genes in HER2-
activated pathways. Based on our stringent criteria for
variant detection, i.e., VAF ≥ 10%, plus other criteria as
described in Additional file1, we identified 27 variants
among 28 samples, representing 21 patients (Fig.5A).
e frequency of PIK3CA mutations among all
sequenced patients was 34% (12/35), similar to that seen
Fig. 4 Molecular Response and Patient Outcomes: NSABP FB‑10. A Kaplan–Meier curves showing association of MR with PFS using a molecular
response cutoff of 50%. B Association between molecular response and best RECIST response
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Jacobsetal. Breast Cancer Research (2024) 26:69
in other studies of unselected metastatic and early-stage
breast cancer patients (cBioPortal). All of the mutations
were in exons 9 and 20 at amino acid 545 and 1,047,
respectively. ese PIK3CA variants also have the highest
VAFs (ranging from 10 to 72% across samples), however,
PIK3CA mutations do not appear to influence patient
outcomes, because response rates between PIK3CA
mutant and WT tumors were similar: 42% (4/12) ver-
sus 45% (14/31), respectively. In one case, a PIK3CA
mutation was detected only in TP1 but this patient had
a PR, again indicating that PIK3CA is not a resistance
marker for study therapy. Variants detected only in PD
or CR patients represent potential resistance or sensi-
tivity markers, respectively, to study therapy. Mutations
detected only in TP1 samples among the 12 paired TP0/
TP1 cases, included ADAM17_S770L, ERBB4_E1010K,
ERBB4_R1040T, and IL6ST_S834* in one sample and
an ESR1_EY537S mutation in another (Fig. 5A). Both
patients had PD, perhaps indicating that these mutations
may have emerged in response to prior therapies. Details
of variants are presented in Additional file1.
Whole transcriptomic proling
We examined the PAM50 subtypes and the 8-gene tras-
tuzumab benefit signature in all available tissues [16].
Among the 29 patients with response and gene expres-
sion data for TP0 tissue, we found that 19/34 (56%)
were HER2E, 8 (24%) were luminal B, 4 (12%) were
basal, 2 (5.9%) were normal, and 1 (2.9%) was luminal
A. Patients with luminal subtype tumors had a lower
CR/PR response rate (1/8 [12.5%]) than patients with
a non-luminal subtype (12/23 [52%]) (Additional file2:
TableS5). Among the TP1 samples with gene expression
data, the frequency of CR/PR was 1/4 in luminal patients
and 5/9 in the non-luminal patients. Intrinsic subtypes
differed between TP0 and TP1 tissues in some cases
(Additional file1: TableS4). Although numbers are small,
the frequency of CR/PR rates were consistently lower
among the luminal patients than non-luminal patients.
e 8-gene trastuzumab signature is a validated sig-
nature for identifying patients with large-, moderate- or
no-benefit from trastuzumab when added to chemother-
apy in the adjuvant setting [16, 17] and has been shown
to associate with pCR rates in the neoadjuvant setting
[21, 22]. We questioned whether this signature may also
show an association with response in the metastatic set-
ting. Among the large-, moderate- and no- benefit groups
the percent of CR/PR patients was 67% (4/6), 50% (9/18),
and 29% (2/7), respectively (data in Additional file 2:
TableS5).
As expected, the level of HER2 RNA increased as the
IHC status increased (i.e., 0, 1 + , 2 + , to 3 +) (Addi-
tional file1: Fig. S2). In TP1 samples they were concord-
ant with patient responses suggesting that HER2 RNA
expression in study entry is associated with response to
T-DM1 + neratinib (Fig.6).
Significant differences were detected in RNA levels
between IHC 1 + and 3 + and between 2 + and 3 + but not
between 0 and 1 + nor between 1 + and 2 + (Additional
file1: Fig. S3). Although numbers are limited, these data
Fig. 5 Variant Alleles in Patients and their Responses: NSABP FB‑10. A Variants detected with a VAF of ≥ 10% in patients with PD, SD, PR or CR.
*indicates a stop codon. B Variant alleles with a VAF of ≥ 5% in patients with PD, SD, PR or CR
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 9 of 13
Jacobsetal. Breast Cancer Research (2024) 26:69
show that the RNA levels are not different between 0
and 1 + . ese patients may benefit from treatment with
other ADCs. e DAISY and DESTINY-Breast04 trials
signal that T-DXd may have significant activity in HER2-
low patients [8, 23].
Discussion
Approximately 35% of HER2-positive patients may have a
loss of HER2 amplification after undergoing chemother-
apy + anti-HER2 therapy [2, 3]. In a retrospective analysis
of 525 patients who received neoadjuvant chemotherapy
(NAC) + HP, 141 patients with residual disease had HER2
status determined pre-and post-NAC-HP. HER2 was
concordant (positive/positive) in 84/141 (60%). HER2
protein expression was lost (IHC 0) in 13/57 (23%) and
designated as HER2-low in 44/57 (77%) including IHC
1 + in 31 and IHC 2 + /FISH non-amplified in 13 [4].
HER2 intratumoral heterogeneity is likely one cause of
discordant HER2 status between primary and post-treat-
ment residual or metastatic disease [24]. Other possibili-
ties include decreased HER2 expression, which could be
a transient change or a result of the selection of HER2-
low subclones [4].
We have assessed HER2 status before and after pre-
and post-study therapy in not only solid tissue but also
blood. We have determined the HER2 status in tissues
with CLIA IHC/FISH assays, which is the gold standard
for HER2 assessment, plus with Ampli-Seq, because it
provided a quantitative analysis of HER2 copy number
with a greater dynamic range. Ampli-Seq was able to
detect a decrease in HER2 copy number in samples that
had not lost HER2 amplification based on IHC/FISH.
We have also monitored HER2 status in liquid biopsies,
which has several advantages over genomic analysis
of tissues. Blood has exposure to all potential meta-
static sites allowing for the detection of different vari-
ants from different metastatic sites. us, blood may
be more representative of the metastatic tumor than
examination of a single biopsied lesion, and may reflect
tumor evolution and intratumoral heterogeneity [25].
Blood samples are more easily collected, making multi-
ple serial collections possible. Collecting multiple serial
tissue samples is impractical, costly, and represents a
much greater risk to patients than does serial collec-
tion of blood. e assessment of ctDNA is a powerful
tool, showing very promising results to monitor tumor
recurrence and response to therapy, but it does not yet
replace the current gold standard, IHC/FISH, for the
assessment of HER2 status in solid tumors. However,
the monitoring of the HER2 status in ctDNA does pro-
vide an indicator of tumor response to therapy.
In our phase Ib/II study, HER2 tissue was amplified
in the baseline samples (TP0) (pre- anti-HER2 therapy)
in all patients by local determination, however, in liq-
uid biopsies at C1D1 after chemotherapy + HP, HE R2-
amplification was detected in only 20/42 (48%) of
patients. Patients with ctHER2-amp versus non-ampli-
fied HER2 ctDNA determined in C1D1 ctDNA had a
longer median PFS, 480days versus 60days (P = 0.015).
It is expected that patients with HER2 amplification
would respond to study therapy (chemotherapy + HP).
Fig. 6 RNA Expression Levels and Response to Therapy. RNA expression levels in TP0 tissues (A) and in TP1 tissues (B) from patients with PD, SD
or CR/PR. The units for RNA expression were log 2 expression values
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 10 of 13
Jacobsetal. Breast Cancer Research (2024) 26:69
Loss of HER2 amplification observed after one cycle
of study therapy may indicate that responders are either
clearing ctDNA completely or that the amplification falls
below the limit of detection. In the 5 cases who were
ctHER2 DNA amplified, and completely cleared ctDNA,
the response rate was 100%.
We applied a Molecular Response VAF ratio calculation
to measure the change in ctDNA from baseline to C2D1,
with the hypothesis that an early decrease in ctDNA lev-
els would predict response to T-DM1 + neratinib ther-
apy, as measured by PFS and RECIST response. Indeed,
Molecular Response was associated with both PFS and
best response to therapy. is should be confirmed in
larger dataset, however, our findings are in line with
other studies demonstrating the ability of ctDNA to pre-
dict short- and long-term efficacy. Early data from the
PADA-1 trial suggests that changing therapy based on
alterations detected in ctDNA, prior to evidence of pro-
gression via imaging, may provide clinical benefit. In
that trial, patients with ER + /HER2-neg ative metastatic
breast cancer being treated in the first line setting with an
aromatase inhibitor (AI) + palbociclib were monitored for
hotspot ESR1 alterations via ctDNA using digital droplet
PCR (ddPCR). Patients with rising ESR1 VAF on therapy,
but no synchronous evidence of disease progression via
RECIST 1.1, were randomized to either continue receiv-
ing an AI + palbociclib or switched to fulvestrant + pal-
bociclib. PADA-1 met its primary efficacy objective, with
patients randomized to receive fulvestrant + palbociclib
having a significantly longer PFS compared to those who
stayed on an AI + palbociclib (median PFS 11.9 months
[95% CI 9.1–13.6 months] versus 5.7 months [95% CI
3.9–7.5months]; stratified HR 0.61 [95% CI 0.43–0.86],
two-sided P = 0.004) [26]. More data on mutational evo-
lution is needed to determine whether similar strate-
gies employing ctDNA to inform change in therapy will
be broadly applicable across breast cancer subtypes and
therapy classes in order to further prolong OS.
Although a cross comparison of studies can be prob-
lematic, phase II and III studies suggest that as patients
are more heavily treated with anti-HER2 regimens, the
PFS and ORR decrease with each subsequent anti-HER2
therapy [27–30]. However, in a phase III randomized trial
of trastuzumab deruxtecan (T-DXd) versus trastuzumab
emtansine (T-DM1) in patients (N = 524) whose disease
progressed on anti-HER2 therapy, the reported ORR for
patients treated with T-DXd or T-DM1 were 79.7% ver-
sus 34.2%, respectively. e landmark analysis of PFS at
12months was 75.8% with T-DXd as compared to 34.1%
with T-DM1 (HR 0.28, 95% CI 0.22–0.37; P < 0.001)
[6]. Although both T-DXd and T-DM1 have a trastu-
zumab backbone, there are substantial differences in
the linker-payload chemistry, which favors an increased
intracellular payload and a bystander effect with T-DXd
[31, 32].
We have shown in our study that the benefit from
T-DM1 + neratinib is limited in HER2-non-amplified
tumors. is finding is consistent with a study reporting
a PFS with T-DM1 of 1.5 months [33] in patients discord-
ant for HER2 in primary and metastatic tissue (HER2-
positive/negative). Although loss of HER2-amplification
appears to be one mechanism of resistance to T-DM1,
half of the patients with HER2-amplified tumors did not
respond to T-DM1 + neratinib, indicating that resistance
to T-DM1 is not limited to loss of HER2-amplification.
Many other mechanisms of resistance to T-DM1 have
been proposed, such as altered cellular uptake, intracel-
lular transport, and metabolism of the payload. [32]
Based on the low level of activity of T-DM1 mono-
therapy in patients failing HP, we speculate that the
combination of T-DM1 and neratinib is more effective
than T-DM1 monotherapy. We are unaware of any tri-
als in HER2-positive breast cancer in which patients
progressing on HP have been randomized to compare
the response to T-DM1 as monotherapy with a com-
bination of T-DM1 and an irreversible tyrosine kinase
inhibitor (TKI). However, in preclinical lung models with
ERBB2 mutation and/or amplification, the combination
of T-DM1 + neratinib did show increased efficacy over
monotherapy. Anecdotally, enhanced efficacy was dem-
onstrated in a breast cancer patient progressing on mon-
otherapy with T-DM1 who then responded with addition
of neratinib. e mechanism of action of the antibody–
drug conjugates (ADC) such as T-DM1 and T-DXd
involves the recognition and binding of the trastuzumab
backbone to the extracellular HER2 surface receptor. e
ADC-protein complex is internalized with cleavage of the
cytotoxic payload. Irreversible TKIs such as neratinib and
afatinib (unlike reversible TKIs such as lapatinib) have
been shown to enhance HER2 internalization and lysoso-
mal sorting, which has the potential to increase uptake of
bound ADC and release of their cytotoxic payload [34].
e KATHERINE [6] study established T-DM1 as a
standard of care in early HER2-positive breast cancer
patients with residual disease after neoadjuvant therapy
[1]. DESTINY-Breast03 has clearly shown superior-
ity of T-DXd over T-DM1 in HER2-positive metastatic
disease. e currently recruiting DESTINY-Breast05
study will compare T-DXd with T-DM1 in high-risk
HER2-positive patients with residual disease following
NAC-HP (NCT04622319). Another study, DESTINY-
Breast06 (NCT04494425), will address the question
of HER2-low (IHC 1 + or IHC 2 + /FISH-negative or
HER2 IHC > 0, < 1 +) in patients with metastatic hor-
mone-positive disease with progression on at least
two lines of endocrine therapy comparing T-DXd with
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 11 of 13
Jacobsetal. Breast Cancer Research (2024) 26:69
investigator’s choice of chemotherapy [31]. e 30-40%
of HER2-positive patients with residual or metastatic
disease after neoadjuvant therapy who have lost HER2
amplification, while not directly being addressed with
these ADCs studies, warrant further investigation with
newer generation ADCs.
Our study has several limitations including the non-
randomized design, the logistic difficulties in obtain-
ing samples of blood and tissue on all patients and
the small sample size, which limited its power and the
ability to perform multivariant analysis. However, the
strengths of our study include the multiple temporal
sample collections, multiple assessments of HER2 sta-
tus, and molecular assessment of DNA in both tissue
and blood.
Despite the limitations of our study, the findings have
generated several hypotheses that should be further
investigated. First, retrospective confirmation in a large
phase III study that loss of HER2-expression under the
pressure of therapy can be detected with liquid biopsy;
second, the overall response, depth, and duration of
response to anti-HER2 therapy is greater in patients
with HER2-amplified than in non-amplified patients;
third, the activity of T-DM1 or other ADCs with a tras-
tuzumab-backbone may be enhanced with addition of
an irreversible TKI such as neratinib. is hypothesis,
testing the interaction of an ADC with a TKI, reversible
and irreversible, could be evaluated in patient-derived
xenografts or other model systems and should be vali-
dated prior to a randomized trial. Finally, a small frac-
tion of HER2-nonamplified patients did benefit from
T-DM1 + neratinib. Possible explanations, which require
further investigation, include a false negative assay,
enhanced internalization of T-DM1 in presence of ner-
atinib, or EGFR becoming the driver in patients with loss
of HER2-amplification, and inhibition by neratinib [32–
34]. We also realize that a low ctDNA fraction could have
prevented the detection of ctHER2 amplification.
Gene expression analysis revealed several important
observations. First, the level of HER2 RNA expression
in TP1 tissues was closely correlated with the response
rate to study therapy. Second, non-luminal subtypes had
a better response rate than luminal tumors, although
this difference was not statistically significant. ird,
there was a non-significant association of the 8-gene
trastuzumab benefit groups with the rate of responses
to study therapy. Fourth, changes in intrinsic subtypes
were seen between TP0 and TP1 tissue samples, indicat-
ing that these changes may be a result of tumors evolv-
ing to become resistant to HP. ese results highlight
the importance of collecting and monitoring molecular
changes in tissue samples as patients move through their
treatments.
PIK3CA mutations are a known oncogenic driver in
breast cancer and drive therapeutic resistance in mul-
tiple HER2-targeted therapies [35]. In the EMILIA
trial, patients with PIK3CA mutations, treated with
capecitabine + lapatinib were associated with a shorter
PFS than were patients with wild-type tumors; how-
ever, in patients treated with T-DM1 this was not the
case [36]. We likewise see that in patients treated with
T-DM1 + neratinib, PIK3CA mutations were not asso-
ciated with outcomes. However, we cannot rule out
the possibility that a subset of patients, refractory to
T-DM1 + neratinib with PIK3CA mutations, may be
responsive to PIK3CA inhibitors.
Conclusions
We demonstrate the usefulness of serial assessment of
HER2 status in blood and tissue in patients with an ini-
tial diagnosis of HER2-positive disease. Loss of HER2
amplification in ctDNA, or the complete loss of ctDNA
on treatment with T-DM1 + neratinib, was associated
with clinical benefit. Further, we show that many of the
patients with short-lived PR or PD were HER2-low in
tissue. ese patients may be better treated with the
recently approved ADC, trastuzumab deruxtecan. We
observed that the ADC, T-DM1, plus neratinib, was
well tolerated. e combination with an irreversible
tyrosine kinase inhibitor with other ADCs warrants
investigation.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13058‑ 024‑ 01823‑8.
Additional le1. Additional Patient information and Methodological
Details.
Additional le2. All Molecular and Response Data Information for NSABP
FB‑10 Patients.
Acknowledgments
We would also like to thank Wendy L. Rea, BA, for editing the manuscript.
Author contributions
Conception &/or Design: SAJ, YW, JA, HF, CL, KPG. Acquisition (of pts/materials)
&/or Analysis: All authors: SAJ, YW, JA, HF, AJM, CL, MF, RCJ, AMS, SKM, SLP, FP,
KQ, KC, RJN, CJA, KV, NW, PCL, AS, KP‑G. Interpretation of the data: SAJ, YW, HF,
FP, KQ, AS, KP‑G. Has drafted the work or substantively revised it: SAJ, YW, AS,
KP‑G.
Funding
We would like to thank our funders BCRF (CONS‑20‑009), Guardant Health Inc.,
Puma Biotechnology, Inc., and the NSABP Foundation. The NSABP Foundation
received funding from Puma Biotechnology to conduct the clinical trial and
for the collection of tissues and blood samples associated with this clinical
trial. No authors received any direct funding for this research, but indirectly
received salary support for efforts to conduct this research. The funder played
no role in the design of the study, or collection, analysis, or interpretation of
the data, or in the writing of the manuscript or submission thereof.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 12 of 13
Jacobsetal. Breast Cancer Research (2024) 26:69
Availability of data and materials
Anonymized individual participant data that underlie the results reported
in this article will be available in dbGAP or other publicly available site after
publication.
Declarations
Ethics approval and consent to participate
Central IRB approval provided by Adverra IRB, Columbia, MD.
Competing interests
AJ Montero: Honoraria: Celgene, AstraZeneca, OncoSec; Consulting/Advisory
Role: New Century Health, Welwaze, Paragon healthcare; Research Funding:
F. Hoffmann‑La Roche Ltd, Basel, Switzerland; Uncompensated: Roche; Open
Payments: https:// openp aymen tsdata. cms. gov/ physi cian/ 618396. K Quinn:
Guardant Health Shareholder. K Chang: Guardant Health Shareholder. RJ Nagy :
Guardant Health Shareholder. PC Lucas: Equity interest in AMGEN outside
the submitted work. KL Pogue‑Geile: Consulting for Bluestar BioAdvisors and
Provisional Patents filed both outside the submitted work. All other authors
declare no other potential conflicts of interest.
Author details
1 NSABP Foundation, Pittsburgh, PA, USA. 2 Cleveland Clinic, Weston/Taussig
Cancer Institute, Cleveland, OH, USA. 3 University Hospitals/Seidman Cancer
Center, Case Western Reserve University, Cleveland, OH, USA. 4 University
of Pittsburgh, Pittsburgh, PA, USA. 5 Present Address: University of Pennsylva‑
nia Perelman School of Medicine, State College, PA, USA. 6 National Institutes
of Health, Washington, DC, USA. 7 Present Address: Virginia Cancer Specialists,
Fairfax, VA, USA. 8 UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 9 University
of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 10 International Drug
Development Institute, Louvain‑la‑Neuve, Belgium. 11 Guardant Health,
Redwood City, CA, USA. 12 University of Florida Health, Gainesville, FL, USA.
13 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,
MN, USA. 14 Autism Impact Fund, Pittsburgh, PA, USA.
Received: 5 October 2023 Accepted: 11 April 2024
Published: 22 April 2024
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