Anatomic Pathology / CELL LINE STANDARD FOR HER-2/NEU ASSAYS
A Formalin-Fixed, Paraffin-Processed Cell Line Standard
for Quality Control of Immunohistochemical Assay of
HER-2/neu Expression in Breast Cancer
Anthony Rhodes, PhD,1 Bharat Jasani, PhD,2 Jérôme Couturier, MD,3
Mark J. McKinley, DipRCPath,4 John M. Morgan, PhD,5 Andrew R. Dodson, FIBMS,6
Hossein Navabi, MSc,7 Keith D. Miller, FIBMS,1 and André J. Balaton, MD8
Key Words: HER-2/neu; Cell line standard; Immunohistochemistry; Fluorescence in situ hybridization; Quality control
Abstract
To ensure the accuracy and reproducibility of
immunohistochemical assays for determining HER2/neu status of patients with breast cancer, a reliable
standard for monitoring assay sensitivity is necessary.
We optimally fixed and paraffin processed human
ovarian and breast carcinoma cell lines SKOV-3, MDAMB-453, BT-20, and MCF-7 in quantities sufficient to
meet the needs of a laboratory for the foreseeable
future. The material was tested, alongside HercepTest
kit cell lines (DAKO, Carpinteria, CA), by 7 breast
cancer centers in the United Kingdom and France with
different immunohistochemical assays and markers. The
cell lines also were analyzed by fluorescence in situ
hybridization (FISH) by 2 centers using HER-2/neu
kits. FISH produced 100% agreement between the 2
centers: SKOV-3 and MDA-MB-453 showed HER-2/neu
amplification and BT-20 and MCF-7 did not.
Immunohistochemical analysis and a common
evaluation method produced 100% agreement that
SKOV-3 and MCF-7 showed 3+ and zero HER-2/neu
overexpression, respectively. For MDA-MB-453, there
was 71% (5/7) concordance of 2+
immunohistochemical staining and 86% (6/7)
concordance of zero or 1+ staining for BT-20.
The cell lines provide a valuable standard for
gauging HER-2/neu assay sensitivity irrespective of the
antibody, antigen retrieval system, detection system, or
method of evaluation used.
© American Society for Clinical Pathology
Clinical trials have shown the potential benefits of
Herceptin (trastuzumab) therapy for patients with invasive
breast carcinomas that overexpress the HER-2/neu protein.1-3
The HER-2/neu gene codes for a membrane surface protein
related to epidermal growth factor receptor. Each of the
surface receptors in this family has a closely associated intracellular tyrosine kinase that is activated when the receptors
bind to their respective ligands, in turn activating other intracellular signals that ultimately are transmitted to the cell
nucleus, resulting in the transcription of genes involved in
controlling cellular replication and differentiation. 4
Trastuzumab therapy, consisting of a humanized monoclonal
antibody, targets the HER-2/neu antigen and inhibits the
growth of HER-2/neu–overexpressing tumor cells. To identify
the 20% to 30% of women with breast cancer that will benefit
most from trastuzumab therapy, a reliable and reproducible
assay is required to detect HER-2/neu overexpression.5-7
Two main test types have evolved: those directed at
detecting amplification of the HER-2/neu gene by fluorescence
in situ hybridization (FISH) and those directed at detecting
overexpression of the HER-2/neu protein by immunohistochemical analysis.4,8-11 Both test types have the advantage of
allowing evaluation of gene amplification (FISH) or protein
overexpression (immunohistochemical analysis) in relation to
tumor morphologic features, unlike molecular techniques that
require homogenization of the tumor.9,12
The main advantages of immunohistochemical analysis
over the FISH method are that it is faster and more economic
to use, and the assay can easily be included as part of a routine
diagnostic service using immunohistochemical analysis, a
service currently offered by virtually all pathology laboratories. The main disadvantages of immunohistochemical assays
Am J Clin Pathol 2002;117:81-89
81
Rhodes et al / CELL LINE STANDARD FOR HER-2/NEU ASSAYS
lie in the variation in the sensitivity of assays between
different laboratories and the different methods of evaluation
frequently used to interpret the results.13-16 This is less of a
problem for some of the routine immunohistochemical
markers that are used merely as adjuncts to histopathologic
interpretation (US Food and Drug Administration Class I
devices) and the results of which require a qualitative rather
than a quantitative assessment.9,17,18 In contrast, for markers
such as those to HER-2/neu and estrogen receptors, the
results of which are analyzed semiquantitatively and suggest
a specific line of treatment to the oncologist or clinician
treating the patient (US Food and Drug Administration Class
II devices), it is vital that the reported index of expression
closely reflect the biological expression of the tumor and is
not influenced adversely by inappropriate assay sensitivity or
the method by which the results are evaluated.
To ensure the accuracy and reproducibility of the results
for HER-2/neu obtained by immunohistochemical analysis, a
standard control is necessary by which day-to-day variation
in the sensitivity of the assay can be accurately monitored. In
this respect, the use of composite tissue blocks representative
of several tumors with varying levels of overexpression for
the HER-2/neu protein is not ideal. Tumor material
frequently is difficult to acquire, and the quantity available
for use for quality control is likely to be limited. Consequently, even during a relatively short time, a laboratory may
need to use several different cases as control specimens, each
with individual variations in tumor expression and fixation.
Obviously this is not ideal for a standard by which to stringently gauge assay sensitivity for HER-2/neu overexpression, as an unpredicted fall or rise in a laboratory’s assay
sensitivity may not be readily detectable when using tumors
of differing expression and fixation as control specimens.
One way around this problem is to use cell lines fixed
and processed in a similar way to histopathologic
specimens.19 Although the characteristics of a cell line are
liable to change with different treatments or cell passages,
large-scale cell production could permit a single harvest of a
sufficient quantity of cells with a specific level of
expression.20,21 Such a harvest of cells of the same phenotype all fixed and processed at the same time would permit a
large and long-lasting bank of standard control material.
Thus, it would have the potential to provide a consistency
over a relatively long period that is not possible with tissuebased control samples.
The aim of the present study was to examine the feasibility of developing a cell line control for HER-2/neu
analysis using 4 cell lines with differing levels of overexpression for HER-2/neu, ranging from unequivocally negative to
unequivocally positive. These cell lines were characterized in
terms of their HER-2/neu gene amplification and protein
overexpression by FISH and immunohistochemical analysis,
82
Am J Clin Pathol 2002;117:81-89
respectively, in several breast cancer centers using different
antibodies and methods. The selected cell lines were optimally fixed and paraffin processed into composite blocks in
sufficient amounts to provide for the needs of a routine laboratory for a protracted period.
Materials and Methods
Cell Lines
Approximately 1 × 108 cells from each of the human
breast carcinoma cell lines, MDA-MB-453, BT-20, and
MCF-7, and the ovarian carcinoma cell line SKOV-3 were
received frozen from the European Collection for Cell
Cultures (CAMR, Salisbury, Wiltshire, England). Samples
of each cell line (3 × 106 cells) for analysis by FISH were
thawed and centrifuged at 1,800 rpm (Sanyo Centaur 2
centrifuge, Sanyo Gallenkamp, Loughborough, England) for
10 minutes. The supernatant was aspirated and the pellet
resuspended in 10 mL of hypotonic solution (0.075-mol/L
concentration of potassium chloride prewarmed to 37°C).
Following incubation for 10 minutes at 37°C, the cells were
centrifuged at 1,800 rpm for 10 minutes, the supernatant
aspirated, and the pellet resuspended in acetic methanol
(methanol/glacial acetic acid, 3:1), with the sequence of
centrifugation and fixation repeated 2 more times. The slide
preparations for FISH analysis were made by placing a
drop of the fixed cell suspension on a clean glass microscope slide and allowing it to air dry and age overnight on
the bench.
The remainder of the cells (9.7 × 10 7 ) were fixed
overnight in 10% phosphate-buffered formalin (pH 7.0) for
approximately 20 hours, embedded in agar, and processed to
paraffin wax, using a method described previously.22 Briefly,
with the fixed cells permitted to sediment in the base of a 25mL polyethylene container with a conical base, the bulk of
the formalin was removed, and the cell suspension removed
to a 1.5-mL microcentrifuge tube and allowed to sediment
again for approximately 30 minutes. The supernatant was
removed, leaving the cells in 10 to 50 µL of fixative, in
which the cells were resuspended. The microcentrifuge tube
containing the cell suspension was heated in a 60°C water
bath to which was added prewarmed and equilibrated 4%
agar. The agar-cell mix then was transferred to 5-mm diameter × 5-mm deep polyethylene molds from which small
agar-cell mix cylinders were extruded carefully. The agarcell mix cylinders were fixed in formalin for another 2 hours
and then placed in a standard tissue-processing cassette and
hand processed to paraffin wax. Cylinders, one from each
cell line, were embedded together in paraffin wax to make
several composite blocks.
© American Society for Clinical Pathology
Fluorescence In Situ Hybridization
FISH analysis was performed and evaluated at 2 centers,
the University Hospital of Wales, Cardiff (M.J.M.), and the
Institut Curie, Paris, France (J.C.). In both centers, a ratio of
more than 2 HER-2/neu/17 centromere probe signals by
FISH was considered to correspond to gene amplification.
The mean number of HER-2/neu signals and chromosome
17 signals per nucleus were calculated by dividing the total
number of signals by the number of nuclei examined.
University Hospital of Wales
The cell preparations were denatured according to the
recommendations in the Vysis PathVysion HER-2 DNA
probe kit (Vysis, Downers Grove, IL), after which they were
hybridized with the Vysis kits HER-2 locus-specific and 17
centromere-specific probes. Subsequent FISH analysis was
performed using 4,6-diamidino-2-phenylindole (DAPI),
fluorescein isothiocyanate (FITC), and Texas Red filters on
the Vysis Smart Capture system. The cells were tested and
screened on 2 separate occasions. Approximately 60 interphase nuclei were screened for each cell line.
Institut Curie
FISH analysis was performed using the Oncor Inform
HER-2/neu (ERBB2) gene amplification detection system
(Ventana Medical Systems, Tucson, AZ) and the instructions
given by the supplier, with slight modification. Briefly, the
preparations were denatured in 70% formamide/2× standard
saline citrate (SSC), pH 7, at 72°C, for 4 minutes. Ten microliters of digoxigenin-labeled HER-2/neu DNA probe were
mixed with 0.5 µL of denatured biotin-labeled chromosome
17 alpha-satellite DNA probe (Oncor, Gaithersburg, MD)
and were applied to the denatured preparations. Slides were
incubated for 15 hours at 37°C; washed in 50%
formamide/2× SSC, pH 7, at 43°C for 15 minutes; and
washed in 2 baths of 2× SSC, pH 7, at 37°C for 4 minutes
each. Detection was achieved using fluorescein-labeled
avidin/antidigoxigenin rhodamine (Oncor). The nuclei were
counterstained using a DAPI/antifade solution. Slides were
viewed with an epifluorescence Leica DMRB microscope
(Leica, Wetzlar, Germany) fitted with a triple band
DAPI/FITC/Texas Red filter. Images were captured using a
Quantics digital camera (Photometrics, Tucson, AZ) and
Quips FISH imaging software (Vysis). A total of 40 interphase nuclei were screened for each cell line.
Immunohistochemical Analysis
Sections were cut at a thickness of 3 µm and mounted
onto slides coated with the adhesive 3-aminopropyltriethoxysilane and dried in a 60°C oven for 4 hours to ensure
maximum adhesion. The position of the cell lines on the
microscope slides, after cutting, is shown in ❚Figure 1❚. Each
© American Society for Clinical Pathology
Slide label
Anatomic Pathology / ORIGINAL ARTICLE
BT-20
MDA-MB-453
MCF-7
SKOV-3
❚Figure 1❚ Position of cell lines on the microscope slide.
circular area of cell line was 4 mm in diameter, and the areas
were 2 mm apart. The approximate surface density for the
cell lines was 800 to 1,100 cells per mm2.
Four sections, along with 2 DAKO cell line controls
(DAKO A/S, Glostrup, Denmark) were sent to collaborating
United Kingdom breast cancer centers in Cardiff, Liverpool,
Manchester, and London and to 1 French center in Bievres,
where they were immunostained with the antibody and
method that each center routinely uses to determine the HER2/neu status of patients with breast carcinoma. This consisted
of a total of 7 immunocytochemical protocols and 4 monoclonal or polyclonal antibodies ❚Table 1❚. The DAKO cell line
controls were immunostained at the same time using the same
antibody and method as used on the new cell line standard.
The immunohistochemical results were evaluated by 3
assessors (A.R., B.J., A.R.D.) scoring independently using a
method of evaluation described previously.1,3,23 Briefly, the
scoring method was as follows: no staining or membrane
staining in less than 10% of tumor cells, 0; faint, barely
perceptible membrane staining in more than 10% of tumor
cells, the cells are stained only in part of their membrane, 1+;
weak to moderate complete membrane staining observed in
more than 10% of tumor cells, 2+; and strong complete
membrane staining in more than 10% of tumor cells, 3+. The
median score from the 3 assessors was used to record the
level of expression for each cell line.
Time Trial to Assess Antigen Decay in Cut Sections
An ongoing time trial to assess the extent of Her-2/neu
antigen decay in the cut sections was implemented with testing
to take place in 2 centers using the CB11 clone and the DAKO
polyclonal antisera at the following times: 0, 2, 4, 8, 12, 24, 36,
and 48 weeks. The immunostaining was conducted in duplicate,
with freshly cut sections acting as the external control samples.
Results
Fluorescence In Situ Hybridization
In both collaborating laboratories using the FISH analysis
and 2 different assays, there was 100% agreement that the cell
lines SKOV-3 and MDA-MB-453 showed HER-2/neu gene
Am J Clin Pathol 2002;117:81-89
83
Rhodes et al / CELL LINE STANDARD FOR HER-2/NEU ASSAYS
❚Table 1❚
Antibodies and Methods Used in Immunohistochemical Analysis of Cell Lines
Study
Code
A
B
C
D
E
F
G
Incubation Antigen
Time
Retrieval
Dilution (min)†
System
Antibody and
Supplier*
Novocastra clone CB11
Novocastra clone CB11
Novocastra clone CB11
Novocastra clone 10A7
DAKO HercepTest‡
DAKO polyclonal
A0485
Oncogene Research
Products clone 3B5
Antigen Retrieval Buffer
Detection
10-mmol/L EDTA, pH 7.0
10-mmol/L citrate, pH 6.0
—
—
DAKO Envision Plus
Vector Elite ABC
Vector Elite ABC
Vector Elite ABC
1:400
1:150
1:160
1:20
40
60
45
45
PC, 2.5 min
PC, 2 min
None
None
1:1,500
25
1:1,500
45
95°C water
5-mmol/L EDTA, pH 7
bath, 45 min
PC, 2 min
10-mmol/L citrate, pH 6.0
Automation
Life Sciences Sequenza
None
A. Menarini Optimax
A. Menarini Optimax
Life Sciences Sequenza
Labeled Streptomyces DAKO Techmate 500
avidin
Streptomyces ABC
None
ABC, avidin-biotin complex; PC, pressure cooker pretreatment.
* DAKO, Ely, Cambridgeshire, England; Life Sciences International, Basingstoke, Hampshire, England; Menarini Diagnostics, Finchampstead, Berkshire, England; Novocastra
Laboratories, Newcastle upon Tyne, England; Oncogene Research Products, CN Biosciences UK, Nottingham, England; Vector Laboratories, Peterborough, England.
† Performed at room temperature unless stated otherwise.
‡ All procedures performed as directed in the HercepTest kit.
amplification and that the cell lines BT-20 and MCF-7 did
not ❚Image 1❚ and ❚Table 2❚.
Immunohistochemical Analysis
The morphologic features and the HER-2/neu expression
of the cell lines are shown in ❚Image 2❚. The results show that
when the expected level of sensitivity was achieved on the
DAKO cell line control (3+, 1+, 0), the sensitivity on the new
SKOV-3
MDA-MB- 453
BT-20
MCF-7
❚Image 1❚ Results of fluorescence in situ hybridization (FISH)
assay for HER-2/neu gene amplification on the cell lines
SKOV-3, MDA-MB-453, BT-20, and MCF-7. FISH analysis
shows the cell lines SKOV-3 and MDA-MB-453 to have HER2/neu gene amplification, while the cell lines BT-20 and MCF7 do not. Red, Oncor Inform HER-2/neu probe (Ventana
Medical Systems, Tucson, AZ); green, Oncor chromosome 17
alpha-satellite probe (Oncor, Gaithersburg, MD).
84
Am J Clin Pathol 2002;117:81-89
cell line control was 3+, 2+, 2+, and 0 with the CB11 clone and
3+, 2+, 1+, and 0 with the HercepTest or the DAKO polyclonal
antibody ❚Table 3❚.
Correlation of Protein Overexpression With Gene
Amplification
For the cell line SKOV-3, there was 100% (7/7) laboratory assay concordance between 3+ HER-2/neu protein overexpression as determined by immunohistochemical analysis
and HER-2/neu gene amplification as determined by FISH.
Similarly, there was 100% concordance between “0” protein
overexpression and no gene amplification for the cell line
MCF-7. For the cell line MDA-MB-453, the level of concordance between 2+ overexpression by immunohistochemical
analysis and gene amplification by FISH was 71% (5/7),
while for BT-20 there was 86% (6/7) concordance between a
“0” or 1+ score by immunohistochemical analysis and no
amplification by FISH ❚Table 4❚.
Time Trial to Assess Antigen Decay in Cut Sections
There was no obvious deterioration of the HER-2/neu
antigen at 2, 4, and 8 weeks when sections of this age were
stained using the Novocastra CB11 clone (Novocastra Laboratories, Newcastle upon Tyne, England) and the DAKO
polyclonal HER-2/neu antibody.
Discussion
Some authors have questioned the reliability of the
immunohistochemical assay for accurately predicting the
likely response of patients to trastuzumab therapy,24-26 while
others have emphasized its reliability, providing that standardized methods with rigorous control of sensitivity are
© American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
❚Table 2❚
Results of Fluorescence In Situ Hybridization Analysis
Total Signals (Mean per Nucleus)
Study Center/Cell Line
No. of Nuclei
Counted
University of Wales Hospital, Cardiff
SKOV-3
MDA-MB-453
BT-20
MCF-7
Institut Curie, Paris, France
SKOV-3
MDA-MB-453
BT-20
MCF-7
HER-2/neu
CEP
HER-2/CEP 17 Ratio
Conclusion
59
60
58
59
571
504
123
123
(9.6)
(8.4)
(2.1)
(2.1)
212
237
182
165
(3.6)
(4.0)
(3.1)
(2.8)
2.7
2.1
0.7
0.8
Amplified
Amplified
Not amplified
Not amplified
40
40
40
40
488
388
76
108
(12.2)
(9.7)
(1.9)
(2.7)
116
128
88
112
(2.9)
(3.2)
(2.2)
(2.8)
4.2
3.0
0.9
1.0
Amplified
Amplified
Not amplified
Not amplified
CEP 17, centromere of chromosome 17 probe.
used.8,27 Such stringent control is not possible without a
graded standard against which laboratory results can be
monitored on a day-to-day basis. Our present study sought to
provide a solution to this problem by developing such a standard using formalin-fixed, paraffin-processed cell lines with
differing levels of overexpression for HER-2/neu.
The use of cell lines to control assay sensitivity for
HER-2/neu is by no means a novel idea, as it is an essential
component of the DAKO HercepTest kit. However, this kit is
relatively expensive, and in the standard kit, only 5 cell
line–based control slides and sufficient reagents to perform
35 tests are provided. Some workers buy the constituent
reagents separately, in which case they are left without the
availability of the valuable control slides, which currently
are not purchasable separately. In addition, other workers
prefer to use different antibodies from the one used in the
HercepTest. The present study provides a solution to this
problem by describing a cell line–based standard by which
assay sensitivity may be accurately gauged, regardless of the
choice of antibody.
To obtain large quantities of cell lines at a standard level
of protein expression, we adopted the approach used by
Ruby and McNally,20,21 using a very large volume of cells
from a single harvest. However, we further extended the
quantity of sections that can be subsequently obtained from
the formalin-fixed, paraffin-processed material by producing
agar cylinders of cells with uniform density22 instead of the
cell pellets used by Ruby and McNally.20,21 We calculated
that 700 to 1,000 sections, of a 3-µm thickness, could be
obtained from one of the blocks that we prepared containing
the 4 cell lines. Thus, just 1 block would provide for control
of a minimum of 700 HER-2/neu assays. Even in the
unlikely scenario of the HER-2/neu assay being performed
by a laboratory daily, 5 days a week, this 1 block would
provide sufficient control material for at least 3 years. As it
is, in the present study, 7 composite blocks were produced,
© American Society for Clinical Pathology
each containing the 4 cell lines with a standard and known
level of HER-2/neu expression, enough to provide even the
busiest of laboratories with sufficient material to last a
minimum of 21 years. Obviously such quantities are unlikely
to be required by 1 laboratory, and, therefore, the process
lends itself to mass-scale production of control blocks by 1
institution for a number of others.
While there is little evidence to suggest that the HER2/neu antigens will decay with time in the formalin-fixed,
paraffin-processed blocks, it is likely that this will occur in the
cut sections.28-31 To study this, we undertook a time trial to
assess the extent of antigen decay in cut sections stored at room
temperature. Although this time trial is ongoing, 8 weeks after
cutting the sections and with testing at 2 centers using the
CB11 clone and the DAKO polyclonal Her-2/neu antibody,
there seemed to be no obvious deterioration in the antigen.
By purchasing the cell lines in the desired volume, it is
possible for a routine histopathology laboratory without cell
culture facilities to devise its own cell line control using the
technology described. At the College of American Pathologists Conference in June 1999 on solid tumor prognostic
factors, it was proposed that “if cell lines are used as standards for HER-2/neu testing, ways to promote production of
cell line controls must be disseminated to practicing pathologists.”32 We hope that the present article assists in this
dissemination by providing details of a relatively simple way
of producing a cell line standard for HER-2/neu.
If reliable, standardized reagent controls are readily
available, the long-standing debate about which antibody,
method, or reagent to use in order to standardize immunohistochemical assays becomes obsolete, because if all assays
are adjusted to achieve an agreed-on end point on an agreedon biological standard, then the method by which this end
point is achieved becomes largely irrelevant.
While the main aim of the present study was to provide
a suitable standard so that assay sensitivity can be gauged
Am J Clin Pathol 2002;117:81-89
85
Rhodes et al / CELL LINE STANDARD FOR HER-2/NEU ASSAYS
A
B
C
D
E
F
86
Am J Clin Pathol 2002;117:81-89
© American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
❚Image 2❚ Results of the HercepTest (DAKO, Ely,
Cambridgeshire, England) for HER-2/neu overexpression on
the cell lines SKOV-3, MDA-MB-453, BT-20, and MCF-7 and
the cell lines from the DAKO HercepTest kit: SK-BR-3, MDA175, and MDA-231. A, 3+ staining of the SKOV-3 cell line
(×400). B, 2+ staining of the MDA-MB-453 cell line (×400).
C, 1+ staining of the BT-20 cell line (×400). D, “0” staining
of the MCF-7 cell line (×400). E, 3+ staining of the SK-BR-3
cell line (×400). F, 1+ staining of the MDA-175 cell line
(×400). G, “0” staining of the MDA-231 cell line (×400).
G
accurately for an important predictive marker, there is the
potential for such an approach to provide an almost limitless
supply of suitable control material for other antibodies. Problems surrounding the use of surgically removed tissues for
quality control, the acquisition of which has become increasingly difficult in the United Kingdom owing to adverse
publicity regarding issues relating to organ retention, also are
partly circumvented if cell lines are used instead.33,34
When the expected level of expression was achieved on
the DAKO cell line controls, the level of expression for the
cell lines SKOV-3, MDA-MB-453, BT-20, and MCF-7 was
found to be 3+, 2+, 1+, and 0, respectively, when using the
HercepTest, or the DAKO polyclonal antisera. Achieving the
expected result on the DAKO cell line–based control with
the CB11 clone resulted in scores of 3+, 2+, 2+, and 0 on
SKOV-3, MDA-MB-453, BT-20, and MCF-7, respectively.
In both of the collaborating laboratories using the FISH
analysis and 2 different assays, there was 100% agreement
that the cell lines SKOV-3 and MDA-MB-453 showed HER2/neu gene amplification and that the cell lines BT-20 and
MCF-7 did not. The discrepancy in the number of signals
between the 2 analytes for the most amplified cell line
(SKOV-3) is probably because the HER-2/neu signals form a
cluster in the nuclei of these cell lines, often making it difficult to recognize them individually and to enumerate them
precisely. Such clusters in interphase nuclei are related to
homogeneously staining regions observed in metaphase
chromosomes (Image 1).
The level of concordance between HER-2/neu gene
amplification determined by FISH and protein overexpression determined by immunohistochemical analysis was
excellent for the cell line showing amplification and high
overexpression (3+; SKOV-3) and the cell lines showing no
amplification and none (0) or little (1+) protein overexpression (MCF-7 and BT-20, respectively). The level of gene amplification as determined by FISH for the cell line MDA-MB-453
❚Table 3❚
Immunohistochemical Staining Characteristics of Cell Lines With Different Antibodies and Methods Used in Participating
Breast Cancer Centers
New Cell Line Control
Study Code Antibody*
A
B
C
D
E
F
G
*
†
CB11
CB11
CB11
10A7
HercepTest
Polyclonal
3B5
DAKO Cell Line Control
SKOV-3
MDA-MB-453
BT-20
MCF-7
SK-BR-3 (3+)†
3
3
3
3
3
3
3
2
2
0
1
2
2
2
2
0
0
0
1
1
0
0
0
0
0
0
0
0
3
3
2
3
3
3
3
MDA-175 (1+)†
MDA-231 (0) †
1
0
0
0
1
1
0
0
0
0
0
0
0
0
See Table 1.
Expected level of staining for a valid result on the DAKO cell line control slide, as given in the HercepTest protocol (DAKO, Ely, Cambridgeshire, England).
© American Society for Clinical Pathology
Am J Clin Pathol 2002;117:81-89
87
Rhodes et al / CELL LINE STANDARD FOR HER-2/NEU ASSAYS
❚Table 4❚
Correlation of HER-2/neu Protein Overexpression by Immunohistochemical Analysis With HER-2/neu Gene Amplification
by FISH
HER-2/neu Status
Protein Overexpression
Cell Line
SKOV-3
MDA-MB-453
BT-20
MCF-7
Gene Amplification
Amplified
Amplified
Not amplified
Not amplified
3+
2+
1+
0
Total
7
0
0
0
0
5
1
0
0
1
2
0
0
1
4
7
7
7
7
7
FISH, fluorescence in situ hybridization.
was judged to be less than that of SKOV-3, though still with
a ratio of greater than 2 for the HER-2/neu/CEP 17 signals.
In the present study, this level of amplification equated to a
2+ level of protein expression with 5 (71%) of 7 of the
immunohistochemical assays used.
The current control has the advantage of providing a
graded series of expression for each of the 3+, 2+, 1+, and 0
categories when using the HercepTest or the DAKO polyclonal antisera. It is appreciated that this method of evaluation may be justifiably criticized for being overly simplistic.5
However, we have used this system because of its use in
previous major publications,1,3 its inclusion in the recommendations by Roche-sponsored United Kingdom referral
centers,23 and because it was the most appropriate scoring
system by which to compare the level of “expected” overexpression exhibited in the DAKO cell lines with that shown
by the new cell lines. We chose the DAKO cell lines from
the HercepTest kit as the point of reference because they are
familiar to most workers in this field, because the HercepTest
is itself undergoing extensive and ongoing validation, and
because of the obvious similarities between the two cell line
control systems. It was not the purpose of the present study
to question methods of evaluation, or indeed which levels of
overexpression most closely predict clinical outcome. Which
level of sensitivity is ultimately found to be most appropriate
and which method of evaluation is used to establish this is
still a matter for debate. However, the value of the cell line
standard control we have described will remain, as it
provides a biological constant against which the variable of
immunohistochemical assay sensitivity for HER-2/neu can
be accurately gauged, regardless of which antibody, antigen
retrieval system, detection system, or method of evaluation
ultimately is used and considered to be appropriate.
From the 1United Kingdom National External Quality Assessment
Scheme for Immunocytochemistry and the Department of
Histopathology, University College London Medical School,
London, England; 2Immunocytochemistry and Molecular
Pathology Unit, University of Wales College of Medicine, Cardiff;
88
Am J Clin Pathol 2002;117:81-89
3Unité
de Cytogénétique Oncologique, Institut Curie, Section
Médicale, Paris, France; 4Haematology Cytogenetics Laboratory,
Department of Haematology, and 5Histopathology Department,
University Hospital of Wales, Cardiff; 6Molecular Pathology and
Development, Department of Pathology, Royal Liverpool
University Hospital, Liverpool, England; 7Department of
Research, Velindre Hospital, Cardiff; and 8Centre de Pathologie,
Bievres, France.
Address reprint requests to Dr Rhodes: Dept of
Histopathology, University College London Medical School,
Rockefeller Bldg, University St, London WC1E 6JJ, England.
Acknowledgments: We are sincerely grateful to Jon Askaa,
PhD, DAKO A/S, Glostrup, Denmark, for providing us with
DAKO cell line control slides and to Elizabeth Anderson, PhD,
Christie Hospital, Manchester, England, and Cheryl Gillett, PhD,
Guy’s Hospital, London, England, for immunostaining cell line
controls.
References
1. Pegram MD, Lipton A, Hayes DF, et al. Phase II study of
receptor-enhanced chemosensitivity using recombinant
humanized anti-p185HER2/neu monoclonal antibody plus
cisplatin in patients with HER2/neu-overexpressing
metastatic breast cancer refractory to chemotherapy
treatment. J Clin Oncol. 1998;16:2659-2671.
2. Slamon D, Leyland-Jones B, Shak S, et al. Addition of
Herceptin (humanized anti-Her2 antibody) to first line
chemotherapy for Her2 overexpressing metastatic breast
cancer (Her2+/MBC) markedly increases anticancer activity:
a randomised multinational controlled phase III trial
[abstract]. Proc Am Soc Clin Oncol. 1998;17:98A. Abstract
377.
3. Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational
study of the efficacy and safety of humanized anti-HER2
monoclonal antibody in women who have HER2overexpressing metastatic breast cancer that has progressed
after chemotherapy for metastatic disease. J Clin Oncol.
1999;17:2639-2648.
4. Wisecarver JL. HER-2/neu testing comes of age [editorial].
Am J Clin Pathol. 1999;111:299-301.
5. Allred DC, Swanson PE. Testing for erbB-2 by
immunohistochemistry in breast cancer. Am J Clin Pathol.
2000;113:171-175.
© American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
6. Slamon DJ, Clark GM, Wong SG, et al. Human breast
cancer: correlation of relapse and survival with amplification
of the HER-2/neu oncogene. Science. 1987;235:177-182.
7. Clark GM. Prognostic and predictive factors. In: Harris JR,
Lippman ME, Morrow M, et al, eds. Diseases of the Breast.
Philadelphia, PA: Lippincott-Raven; 1996:461-485.
8. Jacobs TW, Gown AM, Yaziji H, et al. HER-2/neu protein
expression in breast cancer evaluated by immunohistochemistry: a study of interlaboratory agreement. Am J Clin
Pathol. 2000;113:251-258.
9. Tubbs RR, Stoler MH. The quality of Her-2/neu predictive
immunohistochemistry: something FISHy [editorial]? Mod
Pathol. 2000;13:1-3.
10. Jimenez RE, Wallis T, Tabasczka P, et al. Determination of
Her-2/neu status in breast carcinoma: comparative analysis of
immunohistochemistry and fluorescent in situ hybridisation.
Mod Pathol. 2000;13:37-45.
11. Bankfalvi A, Simon R, Brandt B, et al. Comparative
methodological analysis of erb-2/HER-2 gene dosage,
chromosomal copy number and protein overexpression in
breast carcinoma tissues for diagnostic use. Histopathology.
2000;37:411-419.
12. Kraus MH, Popescu NC, Amsbaugh C, et al. Overexpression
of the EGF receptor–related proto-oncogene erbB-2 in human
mammary tumor cell lines by different molecular
mechanisms. EMBO J. 1987;6:605-610.
13. Rhodes A, Jasani B, Barnes DM, et al. Reliability of
immunohistochemical demonstration of oestrogen receptors
in routine practice: inter-laboratory variance in the sensitivity
of detection and evaluation of scoring systems. J Clin Pathol.
2000;53:125-130.
14. Rhodes A, Jasani B, Balaton AJ, et al. Immunohistochemical
demonstration of oestrogen and progesterone receptors:
correlation of standards achieved on “in house” tumours with
that achieved on external quality assessment material in over
150 laboratories from 26 countries. J Clin Pathol.
2000;53:292-301.
15. Rhodes A, Jasani B, Balaton AJ, et al. Frequency of oestrogen
and progesterone receptor positivity by immunohistochemical
analysis in 7,016 breast carcinomas: correlation with patient
age, assay sensitivity, threshold value and mammographic
screening. J Clin Pathol. 2000;53:688-696.
16. Rhodes A, Jasani B, Balaton AJ, et al. Study of
interlaboratory reliability and reproducibility of estrogen and
progesterone receptor assays in Europe: documentation of
poor reliability and identification of insufficient microwave
antigen retrieval time as a major contributory element of
unreliable assays. Am J Clin Pathol. 2001;115:44-58.
17. Taylor CR. FDA issues final rule for classification of
reclassification of immunochemistry reagents and kits. Am J
Clin Pathol. 1999;111:443-444.
18. Swanson PE. Labels, disclaimers and rules (oh, my!): analytespecific reagents and practice of immunohistochemistry. Am J
Clin Pathol. 1999;111:445-448.
19. Riera J, Simpson JF, Tamayo R, et al. Use of cultured cells as a
control for quantitative immunocytochemical analysis of
estrogen receptor in breast cancer: the Quicgel method. Am J
Clin Pathol. 1999;111;329-335.
© American Society for Clinical Pathology
20. Ruby SG, McNally AC. Quality control of imprint and tissue
section DNA ploidy analysis in image analysis systems
utilizing cell culture–based control materials. Am J Clin
Pathol. 1995;104:167-171.
21. Ruby SG, McNally AC. Quality control of proliferation
marker (MIB1) in image analysis systems utilizing cell
culture–based control materials. Am J Clin Pathol. 1996;106:
634-639.
22. Morgan JM. A protocol for preparing cell suspensions with
formalin fixation and paraffin embedding which minimises
the formation of cell aggregates. J Cell Pathol. 2001;5:171-180.
23. Ellis IO, Dowsett M, Bartlett J, et al. Recommendations for
HER2 testing in the UK. J Clin Pathol. 2000;53:890-892.
24. Jacobs TW, Gown AM, Yaziji H, et al. Specificity of
HercepTest in determining HER-2/neu status of breast cancers
using the United States Food and Drug Administration–
approved scoring system. J Clin Oncol. 1999;17:1983-1987.
25. Roche PR, Ingle JN. Increased HER2 with US Food and Drug
Administration–approved antibody [letter]. J Clin Oncol.
1999;17:434.
26. Pauletti G, Dandekar S, Rong H-M, et al. Assessment of
methods for tissue-based detection of the HER-2/neu
alteration in human breast cancer: a direct comparison of
fluorescence in situ hybridization and immunohistochemistry.
J Clin Oncol. 2000;18:3651-3664.
27. Couturier J, Vincent-Salomon A, Nicolas A, et al. Strong
correlation between results of fluorescent in situ hybridization
and immunohistochemistry for the assessment of the ERBB2
(HER-2/neu) gene status in breast cancer. Mod Pathol.
2000;13:1238-1243.
28. Bertheau P, Cazals-Hatem D, Meignin V, et al. Variability of
immunohistochemical reactivity on stored paraffin slides. J
Clin Pathol. 1998;51:370-374.
29. Wester K, Wahlund E, Sundstrom C, et al. Paraffin section
storage and immunohistochemistry: effects of time,
temperature, fixation and retrieval protocol with emphasis on
p53 protein and MIB1 antigen. Appl Immunohistochem Molecul
Morphol. 2000;8:61-70.
30. van den Broek LJCM, van de Vijver MJ. Assessment of
problems in diagnostic and research immunohistochemistry
associated with epitope instability in stored paraffin sections.
Appl Immunohistochem Molecul Morphol. 2000;8:316-321.
31. Jacobs TW, Prioleau JE, Stillman IE, et al. Loss of tumor
marker–immunostaining intensity on stored paraffin slides of
breast cancer. J Natl Cancer Inst. 1996;88:1054–1059.
32. Hammond MEH, Fitzgibbons PL, Compton CC, et al, for the
Members of the Cancer Committee and Conference
Participants. College of American Pathologists Conference
XXXV: solid tumor prognostic factors: which, how and so
what? summary document and recommendations for
implementation. Arch Pathol Lab Med. 2000;124:958-965.
33. Hunter M. Alder Hey report condemns doctors, management,
and coroner. BMJ. 2001;322:255.
34. Bauchner H, Vinci R. What have we learnt from the Alder
Hey affair? BMJ. 2001;322:309-310.
Am J Clin Pathol 2002;117:81-89
89