Development of automated brightfield double In Situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence In Situ hybridization (FISH)

Article (PDF Available)inDiagnostic Pathology 3(1):41 · November 2008with246 Reads
Impact Factor: 2.60 · DOI: 10.1186/1746-1596-3-41 · Source: PubMed
Abstract
Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas. The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark(R) XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatase (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives. Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 - 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 - 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 - 1.0000). Automated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

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Diagnostic Pathology
Open Access
Methodology
Development of automated brightfield double In Situ hybridization
(BDISH) application for HER2 gene and chromosome 17
centromere (CEN 17) for breast carcinomas and an assay
performance comparison to manual dual color HER2 fluorescence
In Situ hybridization (FISH)
Hiroaki Nitta*
1
, Beatrice Hauss-Wegrzyniak
2
, Megan Lehrkamp
2
,
Adrian E Murillo
2
, Fabien Gaire
2
, Michael Farrell
3
, Eric Walk
1
,
Frederique Penault-Llorca
4
, Masafumi Kurosumi
5
, Manfred Dietel
6
,
Lin Wang
7,8
, Margaret Loftus
7,8
, James Pettay
7,8
, Raymond R Tubbs
7,8
and
Thomas M Grogan
1,9
Address:
1
Office of Medical Affairs, Ventana Medical Systems, Inc, Tucson, AZ, USA,
2
Advanced Staining, Ventana Medical Systems, Inc, Tucson,
AZ, USA,
3
Discovery, Ventana Medical Systems, Inc, Tucson, AZ, USA,
4
Département de Pathologie, Centre Jean Perrin, Clermont-Ferrand Cédex,
France,
5
Pathology and Laboratory Medicine Institute, Saitama Cancer Center, Saitama, Japan,
6
Institute of Pathology, Charité-University Medicine
Berlin, Berlin, Germany,
7
Pathology and Laboratory Medicine Institute, Cleveland Clinic Foundation, Cleveland, OH, USA,
8
The Cleveland Clinic
Lerner College of Medicine, Cleveland, OH, USA and
9
Department of Pathology, College of Medicine, the University of Arizona, Tucson, AZ, USA
Email: Hiroaki Nitta* - hiro.nitta@ventana.roche.com; Beatrice Hauss-Wegrzyniak - beatrice.wegrzyniak@ventana.roche.com;
Megan Lehrkamp - megan.lehrkamp@ventana.roche.com; Adrian E Murillo - adrian.murillo@ventana.roche.com;
Fabien Gaire - fabien.gaire@ventana.roche.com; Michael Farrell - mike.farrell@ventana.roche.com; Eric Walk - eric.walk@ventana.roche.com;
Frederique Penault-Llorca - fpenault@cjp.fr; Masafumi Kurosumi - mkurosumi@cancer-c.pref.saitama.jp;
Manfred Dietel - manfred.dietel@charite.de; Lin Wang - wangle2@ccf.org; Margaret Loftus - loftusm@ccf.org; James Pettay - pettayj@ccf.org;
Raymond R Tubbs - tubbsr@ccf.org; Thomas M Grogan - tom.grogan@ventana.roche.com
* Corresponding author
Abstract
Background: Human epidermal growth factor receptor 2 (HER2) fluorescence in situ
hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab
therapy. However, current HER2 FISH procedures are labor intensive, manual methods that
require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides
cannot be archived for long term storage and review. Our objective was to develop an automated
brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17
centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast
carcinomas.
Methods: The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled
HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark
®
XT slide
processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver
acetate, hydroquinone, and H
2
O
2
reaction with horseradish peroxidase (HRP) and the fast red and
naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity
Published: 22 October 2008
Diagnostic Pathology 2008, 3:41 doi:10.1186/1746-1596-3-41
Received: 29 September 2008
Accepted: 22 October 2008
This article is available from: http://www.diagnosticpathology.org/content/3/1/41
© 2008 Nitta et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified
HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with
94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was
conducted by 4 observers using a conventional brightfield microscope without oil immersion
objectives.
Results: Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated
both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver
dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high
consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases
based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000)
and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa =
0.8993%, 95% CI = 0.8068 – 0.9919) and without the FISH equivocal cases (100%, Simple Kappa =
1.0000%, 95% CI = 1.0000 – 1.0000).
Conclusion: Automated BDISH applications for HER2 and CEN 17 targets were successfully
developed and it might be able to replace manual two-color HER2 FISH methods. The application
also has the potential to be used for other gene targets. The use of BDISH technology allows the
simultaneous analyses of two DNA targets within the context of tissue morphological observation.
Background
The human epidermal growth factor receptor 2 (HER2)
oncogene, located on the long arm of chromosome 17
(17q12-q21), is over-expressed or amplified in approxi-
mately 20% of breast carcinoma cases [1,2]. HER2 status
in breast cancer is used as a prognostic factor, a predictive
factor, and a therapy selection factor [3] for the human-
ized monoclonal antibody trastuzumab (Herceptin
®
;
Genentech), which is an FDA approved drug for use as
monotherapy or combined chemotherapy for treatment
of breast cancer patients with amplified HER2 status. Tras-
tuzumab adjuvant treatment for early HER2 positive
breast cancer is effective for improving patient survival
and cost-effectiveness analyses of such treatment have
shown acceptable ratios [4-7]. However, there is a nega-
tive aspect to trastuzumab therapy, namely cardiac toxic-
ity [3], which is possibly due to myocardial HER2 gene
over-expression associated with anthracycline treatment
[8] and substantial cost.
Quantitative HER2 fluorescence in situ hybridization
(FISH) analyses for detecting HER2 gene amplification
and semi-quantitative HER2 immunohistochemistry
(IHC) analyses for detecting over-expressed HER2 protein
are performed to determine the HER2 status of breast can-
cer patients. The optimal scoring method for determina-
tion of HER2 gene status is the use of chromosome 17
centromere (CEN 17) enumeration for calculating the
HER2/CEN 17 ratio [9]. One study showed that chromo-
some 17 polysomy (13%) and chromosome 17 mono-
somy (2%) were confirmed among 147 breast cancer
cases with 2+ and 3+ HER2 IHC scores [10]. Also, chro-
mosome 17 polysomy is a key prognosis indicator for
breast cancer patients. Patients with chromosome 17
polysomy and no HER2 gene amplification have better
prognosis compared to patients with HER2 gene amplifi-
cation [11]. Dual color FISH for HER2 and CEN 17 targets
is recommended especially for borderline IHC cases [12].
However, there are additional drawbacks to conducting
HER2 FISH assays beyond the requirement for a special-
ized fluorescence microscope and the difficulty of preserv-
ing FISH signal during a long term storage. For example,
HER2 FISH testing has exhibited a higher assay failure rate
in the hands of some investigators when compared to
HER2 IHC testing (5% vs. 0.08%), the FISH assay proce-
dure time is longer than the IHC assay (36 hours vs. 4
hours), and the FISH interpretation time is longer than
IHC interpretation time (7 minutes vs. 45 seconds) [1].
Another disadvantage of the FISH assay is the difficulty of
correlating cytomorphological aspects of the tissue sam-
ple with the gene status [13]. Furthermore, tissue slides for
the dual color FISH test are still processed manually in
most laboratories, which practice can introduce human
errors during the lengthy assay. In fact, FISH assays may
not always be performed accurately [14]. An international
HER2 proficiency testing study showed that there was
20% (4 out of 20 samples) discordance with HER2 FISH
testing among 5 experienced laboratories [15]. On the
other hand, some reports using proficiency testing surveys
conducted by the College of American Pathologists have
demonstrated a much higher concordance for FISH [16].
There are alternatives to FISH for determining HER2 gene
status. The chromogenic in situ hybridization (CISH)
assay using a DAB chromogen and H
2
O
2
substrate system
for horseradish peroxidase (HRP)-based signal detection
has been evaluated and the value of this assay for assess-
ing HER2 status has been demonstrated [12,17-22]. CISH
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slides can be interpreted using an ordinary brightfield
microscope without oil-immersion lenses and can pro-
vide visible tissue morphology for correlation with the
HER2 gene signal. However, with the current CISH
method, the assessment of the HER2/CEN 17 ratio is con-
ducted by enumerating HER2 and CEN 17 separately
using two different tissue sections.
Other brightfield microscopy in situ hybridization (ISH)
methods use autometallography and enzyme metallogra-
phy: 1) Nanogold
®
with gold enhancement in situ hybrid-
ization (GOLDFISH) [23,24] and 2) enzyme
metallography or silver in situ hybridization (SISH) [25-
27]. The GOLDFISH procedure utilizes the tyramide sig-
nal amplification principle and produces large clusters for
amplified HER2 gene signal. On the other hand, the SISH
method produces discrete metallic silver black signals.
Horseradish peroxidase (HRP) of the detection system
reacts with silver acetate, hydroquinone, and H
2
O
2
and
deposits metallic silver particles at the reaction site. The
reaction product can be seen as discrete black dots under
a brightfield microscope. Advantages of SISH include the
high sensitivity for detection of single gene copies, the
high resolution for quantifying DNA targets, and the high
contrast with tissue counterstaining for visual separation
of the signal and tissue morphology [27].
Recently, an automated HER2 SISH assay was evaluated
for assessing the inter-observer interpretative reproduci-
bility of the HER2 gene status of 99 clinical cases when
compared against the reference standard FISH results [28].
Overall concordance between dual color HER2 FISH and
single color HER2 SISH was 96.0% (kappa = 0.754, 95%
CI = 0.518–0.993) and the discrepancies were mainly
observed among tumors with the heterogeneity of tumor
cell populations [28]. Advantages of the SISH assay com-
pared to the CISH assay include that the SISH assay pro-
duces signal clusters and separately visualized discrete
black dots that are easier to count in the majority of clini-
cal samples. With SISH, the endogenous gene copies
present in non-neoplastic stromal cells are also routinely
and reproducibly visualized. However, like the CISH
assay, the detection of CEN 17 signal cannot be performed
on the same tissue section. Thus, it would be ideal to vis-
ualize both HER2 and CEN 17 on the same tissue section
like two-color FISH assays. Dual ISH staining for HER2
gene and CEN 17 would be beneficial for analyzing chro-
mosome 17 aneusomy and for delineation of cases dis-
playing genotypic intratumoral heterogeneity.
One prerequisite for testing HER2 status reproducibly is
the use of automation for conducting the test in the same
manner among different laboratories located in different
parts of the world. Thus, as a step toward standardizing
HER2 testing, our objective was to develop an automated
brightfield double in situ hybridization (BDISH) assay for
simultaneous detection of HER2 and CEN 17 DNA targets
on formalin-fixed, paraffin-embedded breast cancer tissue
samples. Using this method, HER2 status testing can be
conducted in a simplified manner for more accurately
identifying the patients who are eligible for trastuzumab
therapy and potentially leading to the improvement of
breast cancer patient care in the future.
Methods
Tissue samples
MCF7 and BT-474 xenograft tumors were utilized for opti-
mizing the BDISH assay. MCF7 is a breast adenocarci-
noma cell line with non-amplified HER2 status and BT-
474 is a breast ductal carcinoma with amplified HER2 sta-
tus (50–60 copies of HER2) and chromosome 17 polys-
omy [29]. Paraffin sections (4 μm) containing tissue cores
of formalin-fixed, paraffin-embedded MCF7 and BT-474
xenograft tumors were placed onto Superfrost
®
Plus glass
slides (Erie Scientific Company, Portsmouth, New Hamp-
shire).
Ninety-four (94) breast cancer cases were used from the
Cleveland Clinic Foundation and the Cleveland Clinic
Lerner College of Medicine, Cleveland, OH, USA under
IRB approved protocol. Tissue samples were routinely
processed for paraffin-embedding after fixing with an
alcoholic formalin fixative. All breast cancer cases had
been previously tested for HER2 status by FISH using the
PathVysion
®
HER-2 DNA Probe Kit (Abbott Molecular,
Des Plaines, Illinois) at the Cleveland Clinic Foundation.
However, it should be noted that non-consecutive tissue
sections were used for FISH and BDISH analyses.
Brightfield in situ hybridization
The BenchMark
®
XT automated slide processing system
(Ventana Medical Systems, Inc., Tucson, Arizona) was
used for the optimization and performance evaluation of
the BDISH assay for HER2 and CEN 17 DNA targets. A
protocol was established so that the entire assay proce-
dure consisting of baking, deparaffinization, pretreat-
ment, hybridization, stringency wash, signal detection,
and counterstaining was completed as a one-step fully
automated assay. Paraffin tissue sections on glass slides
were baked at 65°C for 20 minutes prior to the deparaffi-
nization step with EZ Prep™ (Ventana) at 75°C for 16
minutes. Deparaffinized tissue sections were pretreated
with a combination of heat treatment with Reaction
Buffer (Tris-based pH 7.6 solution, Ventana) and ISH Pro-
tease 2 or ISH Protease 3 (Ventana) to unmask DNA tar-
gets. Pretreatment conditions were chosen for optimal
signal to noise ratio and tissue morphology preservation
for the xenograft control slides as well as clinical case tis-
sue slides.
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Sequential ISH procedures for HER2 and CEN 17 signal
detection were conducted for a complete BDISH assay
(Figure 1). Reaction Buffer was used for the washing steps
during immunological detection. Liquid Coverslip™ (LCS,
a hydrophobic reagent, Ventana) was used for controlling
liquid evaporation throughout the assay. For HER2 gene
detection, the INFORM
®
HER2 DNA Probe (Ventana), a
dinitrophenyl (DNP)-labeled, nick-translated repeat
deleted DNA probe was applied to the glass slide for co-
denaturing the probe and target at 95°C. Then, the
hybridization step was conducted at 52°C for 2 hours.
After 3 stringency wash steps were performed at 72°C
with 2× SCC (Ventana), tissue sections were incubated
with monoclonal rabbit anti-DNP antibody (Ventana) for
20 minutes and then with HRP-conjugated anti-rabbit
antibody for 16 minutes at 37°C. The metallic silver
deposit for HER2 ISH signal was developed using silver
acetate, hydroquinone, and H
2
O
2
reaction in the presence
of HRP using the ultraView™ SISH Detection Kit (Ven-
tana). For CEN 17 detection, the INFORM Chromosome
17 Probe (Ventana), a DNP-labeled oligoprobe, was
applied to the tissue sections, denatured at 95°C and
hybridized at 44°C for 2 hours. Then, after 3 stringency
wash steps at 59°C with 2× SSC, tissues were incubated
with rabbit monoclonal anti-DNP antibody for 20 min-
utes and then with an alkaline phosphatase (AP)-conju-
gated anti-rabbit antibody for 12 minutes at 37°C.
Finally, the signal for CEN 17 was visualized with a fast
Brightfield double in situ hybridization (BDISH) signal detection scheme with a sequential in situ hybridization methodFigure 1
Brightfield double in situ hybridization (BDISH) signal detection scheme with a sequential in situ hybridization
method. HER2 gene signal was detected with a DNP-labeled nick translated DNA probe hybridization followed by silver signal
detection system (silver acetate, hydroquinone, and H
2
O
2
reaction). Then, chromosome 17 centromere (CEN 17) signal was
detected with a DNP-labeled CEN 17 oligoprobe hybridization followed by fast red and naphthol phosphate reaction signal
detection system.
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red and naphthol phosphate reaction using ultraView Red
ISH Detection Kit. Diaminobenzidine (DAB) chromogen
and H
2
O
2
substrate reagents from the ultraView Universal
DAB Detection Kit (Ventana), 5-bromo-4 chloro-3-
indolyl phosphate (BCIP) substrate and nitro blue tetra-
zolium (NBT) oxidant reagents from the ISH iVIEW™ Blue
Detection Kit (Ventana), and a ready-to-use tetramethyl
benzidine (TMB) solution (Fitzgerald Industries Interna-
tional, Concord, Massachusetts) were also evaluated for
CEN 17 signal detection of the BDISH application.
Because both HER2 and CEN 17 probes were labeled with
the same DNP hapten, CEN 17 signal detection was com-
pleted without DNP-labeled CEN 17 probe after HER2
signal detection to ensure that the anti-DNP antibody of
CEN 17 detection didn't recognize the DNP hapten of
HER2 probe.
Single or double stained tissue sections for HER2 and/or
CEN 17 targets were counterstained with Hematoxylin II
(Ventana) for 4 or 8 minutes and Bluing Reagent (Ven-
tana) for 4 minutes. Counterstained slides were first
rinsed with distilled water containing DAWN
®
(Proctor &
Gamble Company, Cincinnati, Ohio) for removing LCS
from slides and then rinsed with distilled water until soap
was removed completely from the slide. Slides were blot-
ted very gently with paper towels and completely dried at
45°C or 65°C in the oven for at least 15 minutes. One
drop of Cytoseal™ 60 (Richard-Allen Scientific) was
applied onto a dried slide and a glass coverslip was care-
fully placed onto the slide. Excess mounting media was
removed from the slides by gently pressing the slides
against paper towels. Different coverslipping methods
were also evaluated for preserving the fast red staining
during the assay development. BDISH results were
observed with a Nikon ECLIPSE 90i microscope (Nikon
Instruments Inc., Melville, New York) equipped with
Nikon digital camera DXM1200F (Nikon) without oil
immersion objective lenses, up to 60×. However, for pres-
entation purposes, mainly comparing to FISH images
taken with a 100× objective lens, brightfield photographs
contained in this report were obtained using a 100× oil
immersion objective lens.
FISH
PathVysion HER-2 DNA Probe Kit was used for the FISH
test for HER2 and CEN 17 targets of xenograft tumor con-
trols as previously described [30]. Photographs of FISH
images were taken with Zeiss Axioplan 2 microscope (Carl
Zeiss MicroImaging, Inc., Thornwood, New York) with
Metasystems JAIM4+ CCD1 Charge Coupling Imaging
Camera (MetaSystems Group Inc., Watertown, Massachu-
setts) at 100× using an oil-immersion lens.
BDISH performance test
Performance of the BDISH assay was compared to FISH
results as the reference standard using the historical crite-
ria for HER2 amplification with the PathVysion assay
(Negative: HER2/CEN 17 < 2.0 and Positive: HER2/CEN
17 2.0) and using the ASCO/CAP guideline criteria
(Negative: HER2/CEN 17 > 1.8, Equivocal: 1.8 HER2/
CEN 17 2.2, and Positive: HER2/CEN 17 > 2.2) with or
without the equivocal cases. Scoring BDISH slides was
conducted by 4 observers (MK, MD, FPL, and RRT), who
were experienced with scoring HER2 FISH slides. Scoring
occurred at different sites and at different occasions using
different microscopes. Each individual observer evaluated
the set of slides at their own pace and judgement. No
scores were provided by the observers when the staining
quality was deemed not adequate. There was no commu-
nication among observers regarding their scoring experi-
ence of the BDISH slides. Concordance data of FISH
scores vs. consensus BDISH scores among 4 observers and
FISH scores vs. individual BDISH scores by 4 observers
were determined using SAS 9.1 (SAS Institute Inc, Cary,
North Carolina) in calculating frequency tables and
Kappa statistics. The consensus among observers was
defined as the agreement of three or more observers on a
given observation. Scoring of BDISH assays was also ana-
lyzed for the sensitivity and specificity against FISH scores
with the historical scoring method and the ASCO/CAP
scoring method without the equivocal cases. Discordant
cases were investigated by a non-observer (HN) for possi-
ble causes using BDISH slides.
Results
BDISH assay optimization
Images of HER2 single ISH, CEN 17 single ISH, and HER2
and CEN 17 BDISH results with formalin-fixed, paraffin-
embedded xenograft tumor sections are presented in Fig-
ure 2. Single copies of HER2 signal were recognized as
black discrete dots in the nuclei with MCF7 xenograft
tumor (Figure 2A) while amplified HER2 gene signals
were visualized as either an increased number of HER2
signals, clusters of black dots with BT-474 tumor, and/or
both (Figure 2B). Single CEN 17 copies were observed as
red dots that were slightly larger than the black dots for
HER2 genes in the nuclei with MCF7 tumor (Figure 2C)
and BT-474 tumor (Figure 2D). After single staining for
HER2 gene or CEN 17 was optimized, the BDISH applica-
tion with sequential detection for HER2 targets followed
by CEN 17 targets was tested on xenograft tumors. Single
copies of HER2 genes and CEN 17 were stained in the
nuclei of MCF7 tumor cells (Figure 2E) and amplified
HER2 genes and single copies of CEN 17 were visualized
in the nuclei of BT-474 tumor cells (Figure 2F). Because of
the size difference and color contrast of black dots for
HER2 gene and red dots for CEN 17, they could be visu-
ally separated even when red and black signals were co-
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Brightfield in situ hybridization and dual color fluorescence in situ hybridization (FISH) for HER2 and CEN 17Figure 2
Brightfield in situ hybridization and dual color fluorescence in situ hybridization (FISH) for HER2 and CEN 17.
HER2 and CEN 17 detection with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene and
chromosome 17 polysomy) (A, C, E, G) and BT-474 (amplified HER2 gene and chromosome 17 polysomy) (B, D, F, H). Normal
HER2 gene signal is seen as black dots in the nuclei of MCF7 xenograft tumor (A) while amplified HER2 gene signal is seen as
clusters of black dots in the nuclei of BT-474 tumor (B). CEN 17 signal is detected as red dots that are slightly larger than silver
black dots (C, D). Double staining of HER2 gene and CEN 17 is obtained with silver grains and red dots (E, F). Individual HER2
gene and CEN 17 signals can be still recognized when both targets are co-localized (arrow heads, E). HER2 FISH signal is red-
orange and CEN 17 FISH signal is green in the blue nuclei counterstained with DAPI (G, H). 100×.
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localized in the nuclei of MCF7 tumor cells (arrowheads,
Figure 2E). When CEN 17 probe was omitted from the
complete BDISH assay, there was no fast red staining on
xenograft tumor sections (data not shown). Thus, the
anti-DNP antibody used for CEN 17 signal detection (the
second ISH detection) didn't recognize the DNP-hapten
of HER2 probe signal (the first ISH detection) even
though the same hapten was used for the sequential
hybridization method. For image comparison of BDISH
and FISH for HER2 gene and CEN 17, FISH images with
MCF7 tumor and BT-474 tumor are presented in Figure
2G and Figure 2H, respectively. HER2 genes are seen as
red-orange dots and CEN 17 targets are seen as green dots.
One successful way to preserve the fast red staining was
the use of a toluene-based Cytoseal 60 mounting medium
placed onto completely dried tissue sections prior to cov-
erslipping with glass coverslips. We also confirmed that
the red signal was successfully preserved with the Tissue-
Tek
®
film coverslipper method (Sakura Finetek Japan,
Tokyo, Japan) after air-drying slides (data not shown).
The most common method of coverslipping tissue sec-
tions stained with fast red is the use of an aqueous mount-
ing medium. However, this method did not produce crisp
fast red staining for quantitative analyses of BDISH signals
(data not shown). For the second color for the BDISH
application, DAB, BCIP/NBT, and TMB detection systems
that produce brown, blue, and green to blue final product,
respectively, were evaluated. However, they did not pro-
vide sufficient contrast against the HER2 ISH black signal
(data not shown).
BDISH assay performance
After optimizing the BDISH assay for HER2 gene and CEN
17 with formalin-fixed, paraffin-embedded xenograft
tumor sections, we applied the assay to 94 breast carci-
noma cases and scoring BDISH slides was conducted by 4
observers (MK, MD, FPL, and RRT). The consensus among
observers was defined as the agreement of three or more
observers on a given observation. With the historical scor-
ing method (Negative: HER2/CEN 17 < 2 and Positive:
HER2/CEN 17 2.0) (Table 1), the consensus concord-
ance rate was 98.9% (Simple Kappa = 0.9736, 95% CI =
0.9222 – 1.0000), the sensitivity was 96.3%, and the spe-
cificity was 100%. Individual concordance ranges were
between 97.8% (Simple Kappa = 0.9466, 95% CI =
0.8736 – 1.0000) and 100% (Simple Kappa = 01.0000,
95% CI = 1.0000 – 1.0000). With the ASCO/CAP scoring
method (Negative: HER2/CEN 17 > 1.8, Equivocal: 1.8
HER2/CEN 17 2.2, and Positive: HER2/CEN 17 > 2.2)
(Table 2), the consensus concordance rate was 95.7%
(Simple Kappa = 0.8993%, 95% CI = 0.8068 – 0.9919).
Individual concordance ranges were between 92.5%
(Simple Kappa = 0.8275, 95% CI = 0.7102 – 0.9448) and
95.7% (Simple Kappa = 0.9069, 95% CI = 0.8206 –
0.9933). With the ASCO/CAP scoring method without
the FISH equivocal cases (Table 3), the consensus con-
cordance rate was 100% (Simple Kappa = 1.0000%, 95%
CI = 1.0000 – 1.0000). The sensitivity was 100% and the
specificity was also 100%. Individual concordance ranges
were between 97.7% (Simple Kappa = 0.9442, 95% CI =
0.8678 – 1.0000) and 100% (Simple Kappa = 1.0000,
95% CI = 1.0000 – 1.0000).
Representative images of BDISH staining on clinical sam-
ples are presented in Figure 3. Cancer cells were easily
identified based on the tissue morphology and assess-
ments of HER2/CEN 17 ratios could be readily conducted.
Non-amplified HER2 gene cases showed 0–4 copies of
HER2 genes and 0–4 copies of CEN 17 depending on cell
cycle stage and how each cell was cut within a tissue sec-
tion (Figure 3A), while amplified HER2 gene cases
showed multiple copies or clusters of HER2 genes and a
few copies of CEN 17 (Figure 3B). Besides non-amplified
and amplified HER2 cases, cases with a single copy of
HER2 gene due to centromere 17 monosomy or a
monoallelic deletion of HER2 gene (Figure 3C) and mul-
tiple copies of CEN 17 due to chromosome 17 polysomy
(Figure 3D) were also observed.
Table 1: Performance of brightfield double in situ hybridization
(BDISH) with clinical samples based on the historic scoring
method
FISH Total
Positive Negative
BDISH Positive 26 0 26
Negative 16667
Total 27 66 93
Frequency missing = 1
Sensitivity 96.3%
Specificity 100%
Concordance 98.9%
Kappa 0.9736
Table 2: Performance of brightfield double in situ hybridization
(BDISH) with clinical samples based on the ASCO/CAP method
with FISH equivocal cases
FISH Total
Positive Equivocal Negative
BDISH Positive 25 1 0 26
Equivocal 00 00
Negative 0 3 63 66
Total 25 4 63 92
Frequency missing = 2
Concordance 95.7%
Kappa 0.8993
Page 7
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Page 8 of 12
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All discordant cases, that we defined it even by one
observer disagreement between the BDISH and FISH
scores, were re-examined for possible causes of conflicting
results and there were 9 discordant cases. All nine (9) dis-
cordant cases of the BDISH slides presented at least some
degree of the genotypic heterogeneity of tumor cell popu-
lations. In general, there were two types of the tumor cell
heterogeneity with HER2 gene status within the same tis-
sue section: 1) variegated different genotype tumor cell
populations in the same area of tissue section (Figure 4A)
and 2) segregated tumor populations in different areas of
tissue section (Figures 4B&C). Breast cancers with obvious
tumor cell heterogeneity are shown as examples in Figure
4. However, the subtle genotype heterogeneity of tumor
cell populations is often seen among the equivocal cases,
and it also can be seen in Figures 3D &4B which show less
obvious variegated tumor cell heterogeneity. Three (3) of
9 discordant cases demonstrated the segregated tumor cell
heterogeneity while the other 6 cases showed various
degrees of the variegated tumor cell heterogeneity.
Discussion
Accurate HER2 status testing is important for identifying
breast cancer patients who may benefit from receiving
trastuzumab therapy. Currently, in the United States,
HER2 IHC methods are most commonly used for primary
screening for HER2 status, and borderline cases are sub-
jected to dual FISH for HER2 and CEN 17 to determine
the HER2/CEN 17 ratio. Because the discordance rate
between local and central/reference HER2 status testing
with IHC and FISH is significantly high [14,31-33], the
standardization of diagnosing breast cancer cases is recog-
nized as a very important task for improving personalized
cancer patient care [3,34]. The American Society of Clini-
cal Oncology and the College of American Pathologists
has published a guideline recommendation for testing
HER2 status in breast cancer [3] and the Canadian
National Consensus has updated the Canadian HER2/neu
testing guideline [35]. Two potential solutions for
improving the standardization of HER2 status testing
include: 1) automating the entire process for slide staining
[36] and slide reading [36-39] and 2) consolidating the
HER2 testing process within experienced laboratories and
pathologists that perform large numbers of HER2 tests
[15].
One way to improve the accuracy of HER2 status testing is
to automate the assay procedure for HER2 IHC and HER2
FISH assays so that human errors can be diminished.
HER2 IHC assays can be performed using an automated
slide staining system, but HER2 FISH assays remain tech-
nically challenging and time consuming manual molecu-
lar diagnostic assays in most laboratories. An evaluator of
FISH slides must have access to specialized fluorescence
microscopy in a dark room. Because of unstable FISH
staining characteristics, the signals of FISH slides can be
bleached easily, even while reviewing and enumerating
signals. Furthermore, digital images of the FISH slide need
to be captured with a sensitive camera system for each
patient case for the HER2 gene status record. Therefore, it
is desirable to automate a tissue-based HER2 gene status
test that can be observed with a regular brightfield micro-
scope and that produces stained slides that can be
archived.
While the concept of multi-color brightfield ISH applica-
tions was published in 1990's [40,41], it was a recent
achievement to visualize HER2 and CEN 17 targets within
the same nuclei of tissue sections with a manual dual
brightfield ISH application [42]. This dual ISH applica-
tion utilized TMB chromogen for HER2 gene staining.
However, based on published images [28,42], TMB stain-
ing does not provide discrete signals when compared to
the SISH application. The advantages of the BDISH appli-
cation for HER2 gene and CEN 17 presented in the current
study are: 1) the automation of the ISH application; 2) the
visualization of both HER2 gene and CEN 17 targets in
the nuclei of the same cell; 3) the generation of discrete
HER2 gene signals; 4) the ability to reproducibly detect
endogenous HER2 and CEN 17 signals in the stromal tis-
sues and lymphocytes as a reliable internal assay control;
5) the ability to visualise signal with brightfield micros-
copy with non-oil immersion lenses; and 6) the capability
to permanently archive the slides.
HER2 and CEN 17 probes are co-hybridized for dual color
HER2 FISH. However, for the BDISH assay, because the
stringency conditions for the nick-translated HER2 probe
and the CEN 17 oligoprobe were different, it was neces-
sary to conduct sequential ISH staining steps for HER2
gene and CEN 17 targets. For CEN 17 ISH, we have opti-
mized a new detection system with an alkaline phos-
phatase-conjugated antibody and fast red chromogen and
naphthol phosphate substrate reaction. The fast red-based
detection was selected to obtain a good contrast of CEN
Table 3: Performance of brightfield double in situ hybridization
(BDISH) with clinical samples based on the ASCO/CAP method
without 4 FISH equivocal cases
FISH Total
Positive Negative
BDISH Positive 25 0 25
Negative 06363
Total 25 63 88
Frequency missing = 2
Sensitivity 100%
Specificity 100%
Concordance 100%
Kappa 1.0000
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Page 9 of 12
(page number not for citation purposes)
17 ISH signal against the discrete black dots of HER2 SISH
signal. DAB, BCIP/NBT, and TMB detection systems did
not provide sufficient contrast against HER2 ISH black sig-
nal (data not shown). Because fast red precipitate is solu-
ble in organic solvents, in general, aqueous mounting
medium is used for coverslipping. However, the standard
coverslipping method with aqueous mounting medium
on wet tissue sections did not produce tissue sections with
high resolution and therefore detailed tissue structure
could not be observed (data not shown). A successful
method to preserve fast red staining for CEN 17 and high
resolution tissue morphology was, after completely dry
the slides, to apply a toluene-based tissue mounting
medium (Cytoseal 60) for coverslipping with cover glass
or to use a film coverslipper (Tissue-Tek
®
film coverslip-
per). Incomplete drying resulted in faint red background
staining particularly around the fast red precipitate sites
with this method. Interestingly, the use of aqueous
mounting medium onto the dried tissue slides produced
yellowish background staining on tissue sections and this
method did not produce satisfactory results (data not
shown).
The specificity of single ISH for HER2 gene or CEN 17 and
BDISH for both targets was evaluated with xenograft
tumors. HER2 and CEN 17 copy numbers have been doc-
umented previously using the FISH assay [29]. MCF-7
cells are characterized as non-amplified HER2 and chro-
Brightfield double in situ hybridization (BDISH) for HER2 and chromosome 17 centromere (CEN 17) on formalin-fixed, paraf-fin-embedded clinical breast cancer casesFigure 3
Brightfield double in situ hybridization (BDISH) for HER2 and chromosome 17 centromere (CEN 17) on for-
malin-fixed, paraffin-embedded clinical breast cancer cases. Examples of normal HER2 gene (A), amplified HER2 gene
(B), single HER2 gene (C), and chromosome 17 polysomy (D) cases were shown. 100×.
Page 9
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Page 10 of 12
(page number not for citation purposes)
mosome 17 polysomy (3 copies of chromosome 17 per
nucleus) while one of chromosome 17 with HER2 dele-
tion (2 HER2 copies per nucleus) [29]. BT-474 cell line
presents HER2 amplification with 50–60 copies of HER2
genes and 4–6 copies of CEN 17 per nucleus [29]. Ampli-
fied HER2 genes are located not only on chromosome 17,
but also are translocated on other chromosomes [29].
HER2 and CEN 17 copy numbers produced with the sin-
gle target ISH and BDISH methods matched with previ-
ously reported results. As both probes are labeled with the
same DNP hapten, our first concern was to determine if
detecting specific signal for each probe was feasible. We
confirmed that the fast red chromogen detection reagents
did not produce red signal when the fast red ISH was per-
formed without the CEN 17 probe after detection of HER2
by SISH (data not shown). Thus, the SISH detection and
the fast red detection can be combined to perform a
sequential double ISH assay with 2 probes labeled with
the same hapten. Because the sequential BDISH applica-
tion uses 2 specific stringency conditions based on the
length and sequences of 2 probes, it is not necessary to
design 2 probes that require the same stringency for co-
hybridization, like double color FISH assays.
Concordance rates between a set of gold standard dual
color HER2 FISH scores and HER2 and CEN 17 BDISH
scores by 4 observers were calculated for assessing the per-
formance of the BDISH assay. There were 9 discordant
cases (9.6% of the total cases) based on BDISH score dis-
agreement with FISH scores, even by one observer. We
have found that the number of equivocal cases influences
the concordance rate with the ASCO/CAP scoring
method. There were 4 equivocal cases based on FISH
scores and all cases showed the BDISH score disagreement
by at least 2 observers. A similar observation was reported
with an international HER2 testing proficiency study [15].
In their study, the discordant cases (20%) were caused by
the specimen having FISH HER2/CEN 17 ratios between
1.7 and 2.3 that are close to the 'equivocal' defined by
ASCO/CAP HER2 scoring method (1.8 – 2.2). They also
stated "equivocal cases are difficult to interpret, even
highly experienced and validated laboratories" [15]. In
one study, when the FISH assay was used as the primary
test for HER2 status assessment of breast carcinoma cases,
heterogeneity of HER2 gene status was observed in 40 of
742 cases (5%) [43]. It has been speculated that genomic
and phenotypic heterogeneity of tumor cells is the main
reason for the inconsistency of HER2 testing results [44].
The heterogeneity of breast tumor cell populationsFigure 4
The heterogeneity of breast tumor cell populations. The heterogeneity of breast cancer cells was demonstrated with
brightfield double in situ hybridization (BDISH) stained tissue sections. In general, there were 2 types of the tumor cell hetero-
geneity: 1) variegated tumor populations of, as an example, non-amplified (blue asterisk) and amplified (yellow asterisk) HER2
gene cells in the same area (A) and 2) segregated tumor populations of, as an example, discrete (B) and clustered (C) HER2
gene cells in different areas. The tumor cell heterogeneity with different appearance of HER2 and CEN 17 is also seen among
the non-large clustered HER2 cells (B). 100×.
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Page 11 of 12
(page number not for citation purposes)
With current study, all of our discordant cases (9/9 or
100%) displayed the tumor cell population heterogene-
ity: three samples showed significant segregated tumor
cell population heterogeneity (Figures 4B&C) and other
cases showed subtle heterogeneity of tumor cell popula-
tions that are seen among equivocal cases (4/4 or 100%).
Nonetheless, since consecutive tissue sections were not
used for the FISH and BDISH analyses, one can speculate
that the tissue sections for the FISH and BDISH tissue sec-
tions contained tumor cell populations with different
HER2 status. Further clinical evaluations of HER2 and
CEN 17 BDISH application with patient treatment out-
come data are required for more accurate HER2 status
assessment of breast cancer patients to be obtained.
Conclusion
We have successfully developed an automated BDISH
application for HER2 gene and CEN 17 targets in forma-
lin-fixed, paraffin-embedded tissue sections that is highly
concordant to the FISH and is reproducibly interpreted
among observers. Assessment of HER2 gene status can be
conducted without the use of a specialized fluorescence
microscope and the time required for completing HER2
gene status assessment can be shortened significantly. Fur-
thermore, this application has the potential to be used for
other gene targets, any combination of a gene and its chro-
mosome centromere, and tissue section-based gene
assessment tests including gene translocation studies. The
use of BDISH technology allows the simultaneous analy-
ses of two DNA targets within the context of tissue mor-
phology observation.
Competing interests
HN, BHW, ML, AEM, FG, MF, EW, TM are employed by
Ventana Medical Systems, Inc. RRT and MD received grant
support and honorarium for speaking from Ventana Med-
ical Systems, Inc.
Authors' contributions
LW, ML, JP, and RRT were responsible for identifying and
prequalification of the clinical cases used in this study.
HN and TMG were responsible for the BDISH assay devel-
opment and feasibility studies, staining the clinical sam-
ples, and preparing the manuscript draft and image data.
BHW and ML were responsible for the final assay develop-
ment. AEM conducted all statistical analyses for the per-
formance of BDISH assay. FG was the study coordinator
and contributed intellectual content of the study. MF
designed the probes. EW, MK, MD, RRT, and TMG cri-
tiqued the assay performance with their molecular histol-
ogy expertise during the assay development. FPL, MK,
MD, and RRT were the observers for scoring the clinical
samples. All authors contributed intellectual inputs to the
study. All authors read and approved the final manuscript.
Acknowledgements
We would like to thank Vu Nguyen for instrument maintenance and Drs.
Raymond B. Nagle and Guadalupe Manriquez for slide reviewing during the
assay development.
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    • "Clinical examples common in diagnostic hematopathology include detection of KAPPA and LAMBDA mRNA, as discussed above, as well as Epstein Barr Virus Encoded RNAs (EBER). New probe design approaches eliminating any repeat segments, as well as the development of novel haptens and chromogens have opened the possibility for more specific staining with greater sensitivity and the detection of multiple probes per slide [8,9]. Given the advantages of ISH (suitability for fixed tissues , absence of background) as well as technological developments in the field, the current study sought to develop a new methodology known as dual color in situ hybridization (CISH) and to compare its performance inFigure 1 Ig mRNA levels increase with B cell differentiation. "
    [Show abstract] [Hide abstract] ABSTRACT: Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies. The KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator. 39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted; while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant. Optimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856
    Full-text · Article · Jul 2014
    • "As such, they are offered only to those patients for whom there is definitive molecular proof that they harbour the associated specific mutation. Human epidermal growth factor 2 status in breast cancer is one such example and is used as a predictive therapy-selection factor for the humanised monoclonal antibody trastuzumab (Herceptin, Genentech) [110]. Current diagnostic methods, including fluorescent in situ hybridisation and immunohistochemistry , can be subjective and insensitive. "
    [Show abstract] [Hide abstract] ABSTRACT: Reverse transcription quantitative PCR is an established, simple and effective method for RNA measurement. However, technical standardisation challenges combined with frequent insufficient experimental detail render replication of many published findings challenging. Consequently, without adequate consideration of experimental standardisation, such findings may be sufficient for a given publication but cannot be translated to wider clinical application. This article builds on earlier standardisation work and the MIQE guidelines, discussing processes that need consideration for accurate, reproducible analysis when dealing with patient samples. By applying considerations common to the science of measurement (metrology), one can maximise the impact of gene expression studies, increasing the likelihood of their translation to clinical tools. ᅟ
    Full-text · Article · May 2014
    • "For dual-color SISH, 4-lm-thick sections from each microarray block were prepared. The slides were processed using an automated system following the manufacturer's protocols for INFORM HER2 DNA and chromosome 17 (CEP17) probes (Ventana Medical Sys- tem) [10]. Both probes were labeled with dinitrophenol (DNP) and were optimally formulated for use with the ultraView SISH Detection Kit and accessory reagents from the Ventana Benchmark series of automated slide stainers. "
    [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to use immunohistochemistry (IHC) and silver in situ hybridization (SISH) to evaluate alterations in EGFR and HER2 in gastric cancer in order to determine the relationship with prognosis in gastric cancer patients following curative resection. In this study, we analyzed EGFR and HER-2 status by IHC and SISH in 254 stage I-III gastric cancer patients who underwent curative surgery. Thirteen cases (2.48 %) showed EGFR alteration by IHC or SISH. EGFR alteration was associated with older age (P = 0.021), intestinal type (P = 0.040) and higher stage disease (P < 0.001). The patients with operable state gastric cancer who had EGFR alteration had an unfavorable prognosis, and multivariate analysis confirmed that EGFR alteration was an independent unfavorable prognostic factor. Twenty-seven cases (10.6 %) showed HER-2 alteration by IHC or SISH. HER-2 alteration was associated with older age (P = 0.006), well or moderately differentiated histology (P < 0.001) and intestinal type (P = 0.002). HER-2 alteration is not an independent prognostic factor for curatively resectable gastric cancer. We observed EGFR alteration in a subset of cases with operable state gastric cancer and determined that it was associated with an unfavorable prognosis.
    Full-text · Article · Aug 2013
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