Published OnlineFirst March 25, 2014; DOI: 10.1158/1078-0432.CCR-13-2782
Predictive Biomarkers and Personalized Medicine
Clinical
Cancer
Research
Quantitative Measurements of Tumoral p95HER2 Protein
Expression in Metastatic Breast Cancer Patients Treated
with Trastuzumab: Independent Validation of the p95HER2
Clinical Cutoff
Renata Duchnowska1, Jeff Sperinde11, Ahmed Chenna11, Mojgan Haddad11, Agnes Paquet11, Yolanda Lie11,
Jodi M Weidler11, Weidong Huang11, John Winslow11, Tomasz Jankowski2, Bogumiła Czartoryska-Arłukoska-Kłoda5, Barbara Radecka6, Maria M. Litwiniuk4,
wicz3, Piotr J. Wysocki4, Małgorzata Foszczyn
7
8
_
Jolanta Zok
, Michał Wisniewski , Dorota Zuziak9, Wojciech Biernat10, and Jacek Jassem10
Abstract
Purpose: P95HER2 (p95) is a truncated form of the HER2, which lacks the trastuzumab-binding site and
contains a hyperactive kinase domain. Previously, an optimal clinical cutoff of p95 expression for
progression-free survival (PFS) and overall survival (OS) was defined using a quantitative VeraTag assay
(Monogram Biosciences) in a training set of trastuzumab-treated metastatic breast cancer (MBC) patients.
Experimental Design: In the current study, the predictive value of the p95 VeraTag assay cutoff
established in the training set was retrospectively validated for PFS and OS in an independent series of
240 trastuzumab-treated MBC patients from multiple institutions.
Results: In the subset of 190 tumors assessed as HER2-total (H2T)-positive using the quantitative
HERmark assay (Monogram Biosciences), p95 VeraTag values above the predefined cutoff correlated with
shorter PFS (HR ¼ 1.43; P ¼ 0.039) and shorter OS (HR ¼ 1.94; P ¼ 0.0055) where both outcomes were
stratified by hormone receptor status and tumor grade. High p95 expression correlated with shorter PFS
(HR ¼ 2.41; P ¼ 0.0003) and OS (HR ¼ 2.57; P ¼ 0.0025) in the hormone receptor-positive subgroup of
patients (N ¼ 78), but not in the hormone receptor-negative group. In contrast with the quantitative p95
VeraTag measurements, p95 immunohistochemical expression using the same antibody was not significantly correlated with outcomes.
Conclusions: The consistency in the p95 VeraTag cutoff across different cohorts of patients with MBC
treated with trastuzumab justifies additional studies using blinded analyses in larger series of patients. Clin
Cancer Res; 20(10); 1–9. 2014 AACR.
Introduction
P95 was first identified as a band on a HER2 Western blot
analysis, migrating at approximately 95 kilodaltons (1), and
was subsequently shown to be a poor prognostic factor in
HER2-positive breast cancer patients before the era of
Authors' Affiliations: 1Military Institute of Medicine, Warsaw; 2Lublin Oncology Center, Lublin; 3Białystok Oncology Center, Białystok; 4Greater Poland
; 5West Pomeranian Oncology Center, Szczecin;
Cancer Center, Poznan
6
Opole Oncology Center, Opole; 7Warmia and Masuria Oncology Center,
Olsztyn; 8Bydgoszcz Oncology Center, Bydgoszcz; 9Beskidy Oncology
sk, Gdan
sk, Poland;
Center, Bielsko-Biała; 10Medical University of Gdan
and 11Monogram Biosciences, Inc., South San Francisco, California
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Jacek Jassem, Department of Oncology and
sk, ul. D˛ebinki 7, 80-211 Gdan
sk,
Radiotherapy, Medical University of Gdan
Poland. Phone: 48-58-349-22-70; Fax: 48-58 349-22-10; E-mail:
[email protected]
doi: 10.1158/1078-0432.CCR-13-2782
2014 American Association for Cancer Research.
trastuzumab (2). P95 was later shown to be a resistance
marker for trastuzumab (3), a monoclonal antibody used to
treat HER2-positive breast cancer. This is consistent with the
fact that p95 lacks the trastuzumab-binding site, but retains
kinase activity.
More recently, a number of different HER2 carboxy
terminal fragments (CTF) have been identified in breast
cancers, but each of these HER2-CTFs showed strikingly
different activities. The most abundant HER2-CTF, identified as M687-HER2-CTF, seems to be inactive (4). In
contrast, the M611-HER2-CTF form is present in lesser
abundance but is hyperactive, possibly due to its tendency to covalently homodimerize through unpaired
cysteines (4). This form is likely responsible for the
detrimental effects associated with what has become
known as "p95," including potential resistance to trastuzumab (5).
At least two antibodies have been raised against the
M611-HER2-CTF form of p95 (6, 7), as reviewed by Arribas
and colleagues (8, 9). One of these antibodies, D9, has been
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Duchnowska et al.
Translational Relevance
P95 is a hyperactive, truncated form of HER2 that lacks
the binding site for trastuzumab and pertuzumab. It is
variably expressed in HER2-positive breast cancers and is
thought to be a marker for trastuzumab-resistance when
expressed at high levels. A quantitative p95 assay, specific
for the active M611-HER2 carboxy-terminal fragment,
was previously used to identify a p95 cutoff for patients
with metastatic breast cancer (MBC) with poor progression-free survival and overall survival while receiving
trastuzumab-containing therapy. In the current study,
this prespecified p95 cutoff was validated in an independent set of 240 patients with MBC collected from
multiple institutions. The predictive value of p95 was
particularly strong in the hormone receptor-positive
patients. Because p95 does not seem to predict resistance
to HER2 tyrosine kinase inhibitors (TKI), a quantitative
p95 assay may prove useful in identifying patients who
would most benefit from the addition of a HER2 TKI to
therapy containing a HER2-directed antibody or other
anti-HER2 strategies.
used to build a quantitative p95 assay using the VeraTag
platform (6).
In a previous study, an optimal p95 VeraTag cutoff for
progression-free survival (PFS) and overall survival (OS)
was established in a cohort of trastuzumab-treated metastatic breast cancer (MBC) patients (6). The current independent cohort was assembled to validate this prespecified
p95 cutoff.
Materials and Methods
Patient samples
Formalin-fixed, paraffin-embedded (FFPE) primary tumors
from 240 trastuzumab-treated MBC patients were obtained
from the authors’ institutions. Patient characteristics are
shown in Table 1. Trastuzumab was administered on the
basis of the original determination of HER2 positivity as
HER2 IHC 3þ or a HER2/CEP17 FISH ratio 2. Trastuzumab-containing therapy was generally continued until progression, with a median duration on trastuzumab of 10
months. Most patients (84%) received chemotherapy
throughout the entire study period. Estrogen receptor alpha
(ER-a) and progesterone receptor (PR) expressions were
determined by immunohistochemistry (IHC), with 10%
nuclear staining considered positive. Hormone receptor
positivity was defined as either ER-a or PR positivity. This
study was approved by the Ethics Committee of the Medical
University in Gda
nsk (Gda
nsk, Poland), the coordinating
center. All patient data were anonymized before analysis.
Quantitative HER2 assay
Investigators who performed the HERmark and p95
assays were blinded to all clinical and histopathologic data
Table 1. Patient characteristics
Characteristic
Median age at the start of trastuzumab-containing therapy
Stage at presentation
Stage I–III
Stage IV
HER2 level by HERmark
Normal (H2T 13.8)
Overexpression (H2T > 13.8)
FISH/CEP17 (4 unknown)
2.0
>2.0
Tumor grade
Grade 1 (well differentiated)
Grade 2 (moderately differentiated)
Grade 3 (poorly differentiated)
Hormone receptor status
ER-a and PR negative
ER-a or PR positive
Pathology type (8 unknown)
Ductal
Lobular
Other
All patients
HR negative
HR positive
No. (%)
No. (%)
No. (%)
54
55
53
217 (90)
23 (10)
123 (91)
12 (9)
94 (90)
11 (10)
50 (21)
190 (79)
23 (17)
112 (83)
27 (26)
78 (74)
37 (16)
199 (84)
13 (10)
118 (90)
24 (23)
81 (77)
4 (2)
97 (40)
139 (58)
1 (1)
45 (33)
89 (66)
3 (3)
52 (50)
50 (48)
135 (56)
105 (44)
135 (100)
0 (0)
0 (0)
105 (100)
212 (91)
13 (6)
7 (3)
119 (91)
5 (4)
6 (5)
93 (91)
8 (8)
1 (1)
NOTE: HR positivity defined as either ER-a or PR positivity (10% positive nuclear staining).
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Published OnlineFirst March 25, 2014; DOI: 10.1158/1078-0432.CCR-13-2782
P95 Cutoff Confirmation in Trastuzumab-Treated Breast Cancer
Figure 1. A, HERmark HER2 assay (H2T). B, p95 VeraTag assay.
until H2T and p95 results were reported to the Coordinating
Center. Total HER2 protein expression (H2T) was measured
using the HERmark assay (Monogram Biosciences), as
previously described (10, 11). The HERmark assay provides
a tumor averaged HER2 measurement much the same way
HER2 ISH assays are designed to provide a tumor averaged
gene copy number. Briefly, H2T was quantified through the
proximity-based release of a fluorescent tag (vTag for "VeraTag reporter", see Fig. 1A) conjugated via a thioether linker
to a HER2 monoclonal antibody (mAb), Ab-8 (Thermo
Fisher). The antibody was paired with a biotinylated second
HER2 mAb, Ab-15 (Thermo Fisher), linked to a streptavidin
methylene blue photosensitizer molecule (PM). Upon illumination with red light, the PM produced singlet oxygen
(1O2), which cleaved only the thioether linkers in close
proximity, liberating VeraTag reporter molecules. Signal
(vTag), quantified by capillary electrophoresis was normalized to invasive tumor area on the FFPE tissue section.
Multiple cell line controls were included in each batch for
normalization. Analytical cutoffs aligned to central HER2
determination had been determined previously to define
HERmark-negative values (H2T < 10.5 relative fluorescence/mm2 tumor; RF/mm2) and HERmark-positive values
(H2T > 17.8 RF/mm2) with equivocal values defined as 10.5
H2T 17.8. These cutoffs were derived from the <5th
percentile of centrally determined HER2 positives and the
>95th percentile of centrally determined HER2 negatives,
respectively, within a reference database of 1,090 breast
cancer patient samples. The cutoff of 13.8 RF/mm2 that best
discriminated better versus worse patient outcomes on
trastuzumab-based therapy was earlier determined in an
independent cohort of patients (12). This cutoff was used in
all clinical analyses in the current study to define HER2
positivity by the VeraTag assay.
Quantitative p95 assay
The VeraTag p95 assay method and proprietary p95 monoclonal antibody (D9, Monogram Biosciences) characteriza-
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tion was previously described (6). D9 specifically recognizes
the highly active M611-HER2-CTF form of truncated HER2,
but is likely sterically blocked from binding to full-length
HER2 (Fig. 1B). D9 binding to the FFPE tumor section was
detected by a secondary antibody conjugated to a fluorescent
VeraTag reporter molecule. The VeraTag reporter was released
by reduction and quantified by capillary electrophoresis.
Multiple cell line controls were included in each batch for
normalization. Similar to the HERmark assay, relative fluorescence was normalized to tumor area to give units of RF/
mm2. The prespecified p95 2.8 RF/mm2 cutoff, derived
from an independent training set, as previously described
(6), was used to define p95 positivity in the current study.
P95 IHC
p95 expression was assessed by IHC using the same D9
antibody as the p95 VeraTag assay. The p95 immunohistochemical assay was run on a Ventana Discovery XT autostainer using the extended retrieval setting, 2.5 mg/mL D9
anti-p95 and OmniMAP DAB Kit. Four cell line controls
were included in each batch that spanned the range of 0
(negative, no staining) to 3þ (intense staining). p95 IHC
was scored categorically, according to a staining intensity
scale from 0 to 3þ, with at least 10% of the cells staining in
the highest category. P95 IHC H-score was computed as the
sum of 3 [% 3þ] þ 2 [% 2þ] þ 1 [% 1þ].
Statistical analysis
The principal aim of the current study was validation of
the prespecified p95 cutoff of 2.8 RF/mm2 for PFS and OS in
an independent cohort of HER2-positive MBC patients
treated with trastuzumab. The Kaplan–Meier method and
the log-rank test for P values with stratification by hormone
receptor status and tumor grade (1/2 vs. 3) were used for
analysis of PFS and OS. In analyses on hormone receptorpositive or -negative subsets, only tumor grade was used as a
stratification factor. PFS and OS were measured from the
time of initiation of trastuzumab-containing therapy. P95
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Figure 2. Relationship between HER2, p95, grade, and hormone receptor status. A, HERmark H2T versus HER2 FISH/CEP17, with FISH too dense to
2
count indicated as ">30." Solid lines indicate clinical cutoffs at FISH/CEP17 ¼ 2.0 and H2T ¼ 13.8 RF/mm . Dotted lines indicate the equivocal zone
2
2
between HERmark negative (H2T<10.5 RF/mm ) and HERmark positive (H2T>17.8 RF/mm ), as described in the Materials and Methods. B, p95 versus
2
HER2 FISH/CEP17. Those with p95 below the limit of detection are indicated as "<LOD." The p95 clinical cutoff is shown at 2.8 RF/mm . C, p95 versus
H2T with clinical cutoffs described in A and B. Relationship between H2T and clinical variables. D, median H2T is greater in the hormone receptor-negative
subset (P ¼ 0.0047). E, median H2T is greater in the grade 3 subset (P ¼ 0.020). F, median p95 is greater in the hormone receptor-negative subset (P < 0.001).
G, median p95 is greater in the grade 3 subset (P < 0.001).
expression was also assessed by IHC using the same antibody as the p95 VeraTag assay, and correlations with PFS
and OS were calculated using the same methods. The
Mann–Whitney test was used to calculate P values for
differences in distributions of p95 or H2T in grade or
hormone receptor status subgroups.
Results
Relationships between p95 protein, HER2 protein, and
HER2 FISH
HER2 status was originally determined at the local institution by IHC and, for samples showing intermediate
expression (scored 2þ), by FISH. Subsequently, HER2 FISH
status was centrally determined for all but 10 patients. The
FISH HER2/CEP17 ratio and quantitative HER2 expression
(H2T) measurements followed a log–log relationship (Fig.
2A) with a Pearson R2 of 0.45 (P < 1015), after excluding
cases with FISH signals too dense to count, designated as
HER2/CEP17 >30 (Fig. 2A).
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The relationship between VeraTag p95 protein expression
and FISH HER2/CEP17 is shown in Fig. 2B. Samples with p95
below the limit of detection (LOD) were placed at the bottom
of the graph and labeled as <LOD. Tumors with nonamplified HER2 (HER2/CEP17 < 2) expressed low levels of p95,
generally below the prespecified p95 cutoff of 2.8 RF/mm2. In
contrast, 57% of tumors with amplified HER2 were p95 high
(p95 2.8 RF/mm2), with little dependence of p95 level on
the HER2/CEP17 ratio (Pearson R2 ¼ 0.029; P ¼ 0.023).
Overall, the correlation between p95 and H2T (Fig. 2C)
was low (Pearson R2 ¼ 0.20; P < 1010). Expression of p95
was limited at low H2T values, but spanned an approximately 20-fold range at high H2T values. In tumors with
HER2 protein overexpression (H2T 13.8 RF/mm2), 60%
had high p95.
Correlation of p95 and H2T with hormone receptor
status and grade
Both H2T and p95 were negatively correlated with hormone receptor status and positively correlated with tumor
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P95 Cutoff Confirmation in Trastuzumab-Treated Breast Cancer
Table 2. Patient outcomes according to VeraTag p95 status in VeraTag HER-2–positive patients
PFS
Category
N
Univariate analysis
All VeraTag HER2 positivea (n ¼ 190)
VeraTag p95 positivea
114
VeraTag p95 negative
76
Hormone receptor negative (n ¼ 112)
VeraTag p95 positive
80
VeraTag p95 negative
32
Hormone receptor positive (n ¼ 78)
VeraTag p95 positive
34
VeraTag p95 negative
44
Multivariate analysis
VeraTag p95 positive
Hormone receptor positive
HER2 FISH positive
Grade 3
Stage IV
Ductal
Age >55
OS
Median
(mo)
HR (P); p95(þ)
vs. p95(–)
Median
(mo)
HR (P); p95(þ)
vs. p95(–)
7.4
11.9
1.43 (0.039)
1 (1)
28.3
39.6
1.94 (0.0055)
1 (1)
9.7
12.2
0.95 (0.82)
1 (1)
36.0
NR
1.38 (0.34)
1 (1)
6.0
11.1
2.41 (0.0003)
1 (1)
22.4
35.8
2.57 (0.0025)
1 (1)
1.48 (0.027)
1.48 (0.023)
0.50 (0.075)
1.24 (0.20)
1.22 (0.42)
0.84 (0.51)
0.93 (0.64)
1.75 (0.020)
1.80 (0.0092)
0.87 (0.82)
1.10 (0.66)
0.82 (0.57)
1.35 (0.46)
0.82 (0.36)
NOTE: Hormone receptor positivity defined as either ER-a or PR positivity (10% positive nuclear staining). Univariate results including
all patients were stratified by hormone receptor status and tumor grade. Hormone receptor subset analyses were stratified by tumor
grade.
Abbreviation: NR, not reached.
a
VeraTag HER2 positive and p95 positive defined as H2T > 13.8 RF/mm2 and p95 2.8 RF/mm2, respectively.
grade (Fig. 2D–G). Although the correlations were all statistically significant, the differences in the medians were
small, in the range of 1.4-fold to 1.7-fold.
Correlation of p95 with progression-free survival and
overall survival
The primary aim of the current study was validation of
the p95 cutoff at 2.8 RF/mm2, established in a training set
of trastuzumab-treated MBC patients (6). As in the training set, the p95 analysis was restricted to patients with
HER2-positive tumors, as determined by the VeraTag
assay (H2T > 13.8 RF/mm2). Most of the cases that were
HER2 negative by VeraTag were also HER2 negative by
FISH (Fig. 2A).
A p95 cutoff of 2.8 RF/mm2 was established in the
training set as best discriminating the groups of patients
with different PFS and OS. In the current set, the predictive
value of the same cutoff was confirmed for both PFS (HR ¼
1.43; P ¼ 0.039) and OS (HR ¼ 1.94; P ¼ 0.0055; Table 2
and Fig. 3A and B).
Because of the relationship of p95 expression and ER
expression (7), we assessed p95 correlations with PFS and
OS separately for hormone receptor-negative and -positive
subgroups. Among patients with hormone receptor-negative tumors, p95 was not predictive for either PFS (HR ¼
0.95; P ¼ 0.82) or OS (HR ¼ 1.38; P ¼ 0.34; Table 2; Fig. 3C
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and D). Conversely, among patients with hormone receptor-positive tumors, p95 showed strong predictive value for
both PFS (HR ¼ 2.41; P ¼ 0.0003) and OS (HR ¼ 2.57; P ¼
0.0025; Table 2; Fig. 3E and F).
The significance of p95 cutoff was confirmed in multivariable analyses for both PFS (HR ¼ 1.48; P ¼ 0.027) and
OS (HR ¼ 1.75; P ¼ 0.020; Table 2).
P95 IHC
In the current study, 229 of the 240 cases had sufficient
tumor tissue for the p95 immunohistochemical assay. Representative p95 immunohistochemical images are shown
in Fig. 4A and enlarged images in Supplementary Fig. S1.
The results from the p95 VeraTag assay and p95 immunohistochemical assay were in broad agreement, although
within each immunohistochemical category, there was a
range of values from the p95 VeraTag assay (Fig. 4B). The
immunohistochemical analysis using a semiquantitative
H-score showed a correlation (R2 ¼ 0.39; P < 1015)
between these two methods of quantifying p95 (Fig. 4C).
More samples were below the limit of detection in the
immunohistochemical format (29%) than in the VeraTag
p95 format (4.6%).
As with the p95 VeraTag analysis, the p95 immunohistochemical analysis was focused on the cases that were
HER2 positive by VeraTag. Kaplan–Meier plots for the
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Figure 3. Kaplan–Meier plots for PFS (A, C, and E) and OS (B, D, and F) within the HER2 overexpressed subset (H2T>13.8 RF/mm ), split the prespecified
2
2
cutoff into p95 < 2.8 RF/mm (green) and p95 2.8 RF/mm (red). Both receptor subgroups are included in A (P ¼ 0.039) and B (P ¼ 0.0055). C (P ¼ 0.82) and
D (P ¼ 0.34), Kaplan–Meier plots for the hormone receptor (HR)-negative subgroup. E (P ¼ 0.0003) and F (P ¼ 0.0025), Kaplan–Meier plots for the HR-positive
subgroup. P values were calculated by log rank.
p95 immunohistochemical categories are shown in Supplementary Fig. S2A and S2B. In contrast with the p95
VeraTag results, the p95 immunohistochemical categories
did not correlate with either PFS (P ¼ 0.93) or OS (P ¼
0.54). Cutoffs between each immunohistochemical level
were also tested, but none gave a statistically significant
result for PFS or OS. Continuous p95 H-score and IHC 0/1þ
versus 3þ were also tested with the same result.
With the VeraTag p95 assay, the strongest correlations
with PFS and OS were seen in the hormone receptorpositive subset. P95 IHC in this group showed a trend with
shorter PFS (P ¼ 0.071) and no correlation with OS (P ¼
0.96; Supplementary Fig. S2C).
Discussion
Since the discovery of a truncated form of HER2 as a
potential biomarker of trastuzumab resistance, the molecule commonly called p95HER2 or p95 has been measured
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and defined in a number of different ways, each capturing a
different subset of the mixture of active and inactive members of the HER2-CTFs family (4). Not surprisingly,
attempts to infer active p95 levels by comparing HER2
intracellular and extracellular domain content have led to
inconsistent results (13). Even if the expression of various
HER2-CTFs were correlated to some degree, obtaining an
accurate measure of the minor component of active HER2CTF would be plagued by a high degree of variability. This
situation prompted the development of specific antibodies
for the M611-HER2-CTF (6, 8).
Using the M611-HER2-CTF–specific D9 antibody in a
VeraTag assay, an optimal clinical cutoff for poor outcome
in trastuzumab-treated MBC patients was established in a
training set (6) and verified in the current test set. As a
marker for poor outcome of trastuzumab, high p95 expression may identify tumors that are susceptible to a HER2
tyrosine kinase inhibitor (TKI) used alone or in combination with trastuzumab-containing therapy (14). Upcoming
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P95 Cutoff Confirmation in Trastuzumab-Treated Breast Cancer
Figure 4. P95 IHC results. A, typical p95 immunohistochemical staining for each category. B, relationship between p95 VeraTag and p95 IHC category.
C, relationship between p95 VeraTag and p95 IHC H-score.
clinical studies comparing trastuzumab with trastuzumab
in addition to a HER2 TKI, with prospective p95 assessment,
may verify this hypothesis.
In this study, surprisingly the hormone receptor-positive cases seemed to be the major driver of the correlation between p95 and outcomes. This may be unique to
the metastatic setting, where most patients with hormone
receptor-positive disease had already progressed on
endocrine therapy in the adjuvant setting. Some of this
resistance to endocrine therapy may have been driven by
p95. The observed relationship between p95 levels and
hormone receptor status is consistent with a previous
finding (7). The reasons why the hormone receptorpositive set would drive this correlation are not well
understood, as the characteristics seemed to be well
distributed between hormone receptor-negative and
-positive groups (Table 1) except for a higher likelihood
for hormone receptor-negative tumors to be grade 3 (P ¼
0.0056). A recent report has revealed that p95 expression
can induce a senescence secretory phenotype that can
support metastasis of proliferating breast cancer cells (5).
It could be hypothesized that with continued p95 expression in metastatic lesions, as we have observed (15), p95induced protumorigenic factors may promote a more
aggressive phenotype. However, it is unclear why this
would be enhanced in the hormone receptor-positive
subset.
At first glance, the percentage of HER2-positive tumors
with p95 expression above the prespecified p95 cutoff may
seem higher than expected. However, earlier reports on
rates of p95 positivity were based an analytical cutoffs that
may have been limited by the sensitivity of the methods
used (1, 2). As shown in Fig. 2, there is no obvious
analytical break point that could be used to define p95
positivity. The p95 VeraTag cutoff was set in the training set
(6) based on clinical outcomes. The training set had a lower
percentage of cases above the p95 cutoff, but was also
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composed of a population with lower H2T and FISH
HER2/CEP17 distributions.
VeraTag was chosen as the preferred format due to
increased sensitivity, quantitative output, and to avoid the
subjective quantification that is required for IHC. Other
quantitative approaches may give a readout more similar to
VeraTag, such as visualization of immunofluorescence
directly on the slide versus capillary electrophoresis quantification of the cleaved fluorescent tag used in the VeraTag
method. IHC was chosen here as a point of comparison
because it is the most commonly used method for assessing
protein expression in FFPE samples. The p95 immunohistochemical assay was optimized for specific p95 detection in
the presence of HER2 and maximal staining in positive
cases without appreciable background in negative cases.
Although it is conceivable that a different staining protocol
could have yielded somewhat different results, we believe
that the data presented here are a valid comparison of p95
by VeraTag and p95 IHC. In the current cohort, there was a
broad correlation between VeraTag assay and IHC using the
same antibody (Fig. 4C; R2 ¼ 0.39; P < 1015); however,
IHC did not distinguish favorable from poor outcomes.
Some of this variation likely comes from the method of
quantification. As is customary with IHC scoring, the immunohistochemical category was determined by the staining
level of the most intensely stained cells, in this case the top
10%. In contrast, the VeraTag assay provides a measure of
the tumor averaged p95 expression. Therefore, more heterogeneous staining could give a higher immunohistochemical category than homogeneous staining at the same
p95 VeraTag level. Nonetheless, IHC may still prove useful
in detecting heterogeneous expression of p95 within the
tumor, should this prove to be important. The correlation of
treatment efficacy outcomes with p95 VeraTag values and
the lack of correlation with p95 IHC suggest that a tumoraveraged measurement of p95 expression using the VeraTag
method may be more clinically useful.
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Duchnowska et al.
Recently, a different p95 immunohistochemical assay
was assessed in patients administered neoadjuvant trastuzumab in the GeparQuattro study (16). This assay used a
different antibody and at a concentration high enough to
give 82% p95 positivity, using a cutoff of 20% strong
staining, among those that were HER2 positive by silver in
situ hybridization. Surprisingly, p95 positivity at this cutoff
correlated with a higher pathologic complete response
(pCR) rate. It is not known whether inadvertent measurement of full-length HER2 or a difference in setting contributed to this result. In support of the former point, HER2
mRNA levels have been found to correlate with increasing
pCR in GeparQuattro at least in the ESR1-positive tumors
(17). A recent report of the VeraTag p95 assay in the
NeoALTTO clinical trial (18) also supports the notion that
p95 expression in the neoadjuvant setting may have different significance than in the metastatic setting investigated
here. Although pCR generally correlates with longer term
outcomes, this is not the case in luminal B/HER2-positive–
like tumors (19), the subgroup whose outcomes were most
correlated with p95 expression in the current metastatic
study.
Even though our study is among the few examples of
positive validation of a novel predictive marker in advanced
breast cancer, we are aware of its limitations. First, it was
retrospective, although the p95 cutoff was prespecified.
Second, this study used measurements on primary tumors
to predict outcomes in the metastatic setting, whereas some
tumor characteristics might have changed during progression. We have compared the VeraTag assay with p95 IHC
rather than with a more quantitative method such as
immunofluorescence that might yield similar results to
VeraTag. Finally, as with any tissue-based immunoassay,
preanalytic sample processing might have impacted measurements in FFPE tissue. Some of these factors may have
resulted in the relatively modest PFS difference observed in
the p95 subgroups (HR ¼ 1.43). The difference was greater
in the training set (HR ¼ 1.9; ref. 6), as expected for an
optimized cutoff. The OS results were more similar in the
current (HR ¼ 1.94) and training (HR ¼ 2.2; ref. 6) sets.
In conclusion, a clinical VeraTag p95 cutoff derived from
a previous training dataset was confirmed in a second
independent clinical series as a predictor of clinical outcomes on trastuzumab. The observed consistency in the p95
VeraTag cutoff across different cohorts of patients with MBC
treated with trastuzumab justifies additional studies using
blinded analyses in larger series of patients. Clinical relevance of quantitative p95 protein expression remains to be
established in a controlled clinical trial.
Disclosure of Potential Conflicts of Interest
J. Sperinde is employed as a director/scientist and has ownership interest
(including patents) in LabCorp. A. Chenna is employed as a scientist and has
ownership interest (including patents) in Labcorp. A. Paquet has ownership
interest (including patents) in Labcorp stock. J.M. Weidler is employed in
Monogram Biosciences/LabCorp. W. Huang is employed as a senior director,
Clinical Research in Monogram Biosciences/Integrated Oncology-LabCorp.
J. Winslow is employed as director, Oncology in Monogram Biosciences/
LabCorp, Inc. No potential conflicts of interest were disclosed by the other
authors.
Authors' Contributions
Conception and design: R. Duchnowska, J. Sperinde, J.M. Weidler,
W. Huang, J. Jassem
Development of methodology: R. Duchnowska, J. Sperinde, A. Chenna,
W. Huang, J. Jassem
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): R. Duchnowska, J. Sperinde, W. Huang, J. Winslow, T. Jankowski, B. Czartoryska-Arłukowicz, P.J. Wysocki, M. Foszczy
nska-Kłoda, B. Radecka, M.M. Litwiniuk, M. Wisniewski, D. Zuziak,
W. Biernat
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Duchnowska, J. Sperinde, M. Haddad,
A. Paquet, J.M. Weidler, W. Huang, J. Jassem
Writing, review, and/or revision of the manuscript: R. Duchnowska,
J. Sperinde, A. Chenna, M. Haddad, A. Paquet, Y. Lie, J.M. Weidler, W. Huang,
J. Winslow, T. Jankowski, B. Czartoryska-Arłukowicz, B. Radecka, D. Zuziak,
W. Biernat, J. Jassem
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Duchnowska, J. Sperinde, Y. Lie,
_
W. Huang, T. Jankowski, B. Czartoryska-Arłukowicz, B. Radecka, J. Zok,
D. Zuziak
Study supervision: R. Duchnowska, J. Winslow
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received October 9, 2013; revised February 17, 2014; accepted February
17, 2014; published OnlineFirst March 25, 2014.
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Published OnlineFirst March 25, 2014; DOI: 10.1158/1078-0432.CCR-13-2782
Quantitative Measurements of Tumoral p95HER2 Protein
Expression in Metastatic Breast Cancer Patients Treated
with Trastuzumab: Independent Validation of the p95HER2
Clinical Cutoff
Renata Duchnowska, Jeff Sperinde, Ahmed Chenna, et al.
Clin Cancer Res Published OnlineFirst March 25, 2014.
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