Anatomic Pathology / FISH for HER2: When to Use Chromosome 17
Determination of HER2 Amplification by In Situ
Hybridization
When Should Chromosome 17 Also Be Determined?
John M.S. Bartlett, PhD, FRCPath,1 Fiona M. Campbell, MSc,1 and Elizabeth A. Mallon, MD2
Key Words: HER2; Breast; Fluorescence in situ hybridization; FISH; In situ hybridization; Amplification; Copy number; Chromosome 17;
Aneusomy; Trastuzumab
DOI: 10.1309/AJCPSDG53BEANCYE
Abstract
Our purpose was to determine the accuracy
of diagnosis of HER2 amplification by analysis of
HER2 copy number. HER2 and chromosome 17
were measured by dual-color fluorescence in situ
hybridization in breast cancer samples. At a HER2
copy number of 2 to less than 3, 16 (3.3%) of 488 cases
had HER2 amplification; and at a copy number of 3
to less than 4, 32 (16.4%) of 195 cases were amplified.
The proportion of cases with HER2 amplification
increased considerably at HER2 copy numbers of 4
to less than 7: 50.0% at 4 to less than 5; 67.5% at 5 to
less than 6, and 77.3% at 6 to less than 7. Virtually all
cases were amplified at HER2 copy numbers of 7 or
more. We recommend that all breast cancer cases with
a HER2 copy number of 2 to 7 determined by singlecolor in situ hybridization should also be analyzed for
chromosome 17 to obtain a more accurate diagnosis
of HER2 amplification.
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Accurate diagnostic testing of HER2 expression and/
or gene amplification is an important aspect of breast cancer
management and is required for treatment decisions in breast
cancer, in particular relating to treatment with trastuzumab
(Herceptin).1-4 Guidelines for breast cancer management in
the United Kingdom, European Union, United States, and
many other countries require that all patients with breast cancer be tested accurately for HER2 status at initial diagnosis or
at the time of recurrence.1,2 No rationale for excluding patients
from HER2 testing has been established.5 Establishing tumor
HER2 status may also be of value for guidance in predicting
responses to other agents such as tamoxifen, taxanes, and
anthracyclines.2-5
Amplification of the HER2 gene drives overexpression
of the oncoprotein6,7; however, the majority of laboratories
rely on a 2-tier testing procedure of immunohistochemical analysis followed by fluorescence in situ hybridization (FISH) for equivocal (predominantly with an immunohistochemical score of 2+) cases, according to UK, US,
and Canadian guidelines.1,2,8-11 Good correlation between
immunohistochemical analysis and FISH for HER2– tumors
(immunohistochemical score of 0 or 1+; no amplification) and
for tumors with high levels of HER2 overexpression (immunohistochemical score of 3+; 90%-95% amplification) can be
achieved with care, but the performance of FISH on the population with immunohistochemical scores of 2+ is essential for
accurate identification of HER2+ tumors.12
Previous studies have shown that FISH is the most accurate, reproducible, and precise predictor of HER2 overexpression in routine diagnostic laboratories.6,7,13 Molecular testing
based on in situ hybridization (ISH) is increasingly regarded as
more predictive of response to trastuzumab and other agents.3
© American Society for Clinical Pathology
Anatomic Pathology / Original Article
ISH tests measure HER2 copy number using FISH or chromogenic ISH (CISH) detection methods.1,14 Dual-probe FISH
or CISH, with HER2 and chromosome 17 probes, determines
the ratio of the HER2 gene to the chromosome 17 copy number
and provides an accurate means of assessing gene amplification
(an increase in copies of HER2 per chromosome) rather than
increases in copy number due to chromosomal duplication.
Aberrations of chromosome 17 (aneusomy) are common
in breast cancer15 and, therefore, have a critical impact on the
assessment and reporting of HER2 gene amplification in a
significant subset of cases. There is an ongoing debate as to the
proportion of cases for which assessment of chromosome 17
copy number may be important. This debate is centered around
2 questions: first, the frequency of aneusomy of chromosome
17 in breast cancer, and second, the impact of such aneusomy
on diagnosis of gene amplification. Misunderstandings relating
to the determination of chromosomal copy number in tissue sections underpin the majority of false assumptions made regarding the frequency of aneusomy of chromosome 17 in breast
cancer and, therefore, the impact this aberration has on the
diagnosis of HER2 gene amplification. These misunderstandings have led to widespread inappropriate use of single-color
ISH techniques for testing HER2 “gene amplification,” which
rely on the false assumption that aneusomy of chromosome 17
is infrequent and rarely impacts on the diagnosis of HER2 gene
amplification. The purpose of this article is to provide data to
refute this assumption and to provide advice on the inclusion of
chromosome 17 copy number testing for laboratories performing diagnostic testing for HER2 gene amplification.
According to current guidelines,1,2 amplification of HER2
is considered to be a HER2/chromosome 17 ratio of 2.0 or
more, and a ratio less than 2.0 is regarded as nonamplified. For
HER2 gene copy number assays, it has been assumed that copy
numbers of more than 6.0 reflect amplification, and results of
less than 4.0 HER2 gene copies per nucleus is always associated with lack of amplification; cases with 4 to 6 copies per cell
are thought to require validation by testing of a parallel section
for chromosome 17.1,2 To our knowledge, this assumption has
not been verified experimentally. If cases with fewer than 4
observed copies of HER2 per cell have fewer than 2 copies
of chromosome 17 per cell, they may be amplified. Similarly,
cases with 6 or more HER2 copies may be nonamplified if
they exhibit more than 3 copies of chromosome 17. Hitherto,
the assumption has been made that such cases do not exist.
If this assumption is incorrect, a proportion of cases will be
incorrectly diagnosed.
Previous data from our group have shown that owing to
nuclear truncation in thin tissue sections and incomplete hybridization of ISH probes in diagnostic specimens, the observed
mean chromosomal copy number (MCCN) falls significantly
below the theoretical value of 2 copies per cell. By using
normal breast tissues, Watters et al15 have demonstrated that
© American Society for Clinical Pathology
the observed MCCN for chromosome 17 in diagnostic tissue
sections is 1.61 ± 0.08 (mean ± SD). By using this value, a
normal range for disomy 17 can be calculated using the 99%
confidence intervals for the mean value; the calculated range
is, thus, 1.35 to 1.85 observed copies per cell. Observed values
from diagnostic breast cancer specimens beyond this range
must, therefore, reflect monosomy (values <1.35) or polysomy
(values >1.85). This principle, which has been validated by
using other tissues and chromosomal probes, underpins much
of the work on detection of chromosomal aneusomy by ISH
methods.16-19 By using the definition of 1.35 to 1.85 copies
per cell for disomy, Watters et al15 have shown that more than
50% of breast cancers exhibit chromosome 17 aneusomy,
which may result in a higher copy number of HER2, owing to
chromosome 17 polysomy, in the absence of HER2 amplification. These observations have been repeated by others (verbal
communication, Kirsten van Neilsen, PhD, DAKO, Glostrup,
Denmark, December 2007), but the implication of this high
rate of chromosomal aneusomy for single-color ISH assays has
not been explored. However, one immediate consequence can
be predicted: because the observed MCCN for chromosome
17 falls within the range of 1.35 to 1.85 copies, a tumor with
an observed mean HER2 copy number of 3 and an observed
MCCN of 1.4 for chromosome 17 would, in fact, be amplified.
Under current guidelines, however, there is no requirement
for these “low copy” tumors to be tested for chromosome 17.
Therefore, measurement of the chromosome 17 copy number in
conjunction with HER2 gene copy number is critically important for the accurate diagnosis of HER2 gene amplification.
The aim of this study was to analyze the frequency of
HER2 amplification (determined by dual-color FISH) relative
to the HER2 copy number in a large population of breast cancer patients to determine the accuracy of diagnosis of HER2
amplification as determined by using current guidelines. We
provide evidence to support the requirement for measurement
of chromosome 17 for a broader range of HER2 copy number
determined by single-color ISH to obtain a more accurate
diagnosis of HER2 amplification. We also highlight the significant risk of misdiagnosis of HER2 gene amplification by
using current guidelines for single-probe HER2 assays.
Materials and Methods
Study Cases
In an audit of HER2 testing, HER2 and chromosome 17
were measured by FISH in breast cancer samples referred
between 2000 and 2008 from external hospitals to 1 author’s
(J.M.S.B.) laboratory, an established UK reference laboratory.
Data on pathologic type, grade, and other such variables are
not included because these were not required for referral of
HER2 cases from external sources.
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Determination of HER2 by Immunohistochemical Analysis
HER2 was determined by immunohistochemical analysis
using the HercepTest (DakoCytomation, Carpinteria, CA),
as previously described.7 The HercepTest scoring protocol
assigns overall HER2 scores of 1+, 2+, or 3+ if 10% or more
of carcinoma cells attain weak, moderate, or intense HER2
membrane staining, respectively.
Determination of HER2 and Chromosome 17 by FISH
HER2 and chromosome 17 were determined by dual-color FISH using the PathVysion detection kit (Vysis, Downers
Grove, IL), as previously described.7 This is an established
method used routinely for clinical diagnosis.1,8 FISH-stained
sections were scanned at ×1,000 magnification, and 3 separate
carcinoma areas identified. The numbers of chromosome 17
and HER2 signals were counted in 20 to 60 nonoverlapping
nuclei using at least 3 distinct tumor fields, or more if there
was heterogeneity. The mean HER2/chromosome 17 copy
ratio was calculated, and the mean HER2 and mean chromosome 17 copy numbers observed were recorded. For the
purposes of this audit, the mean HER2 copy number from
the PathVysion system was used to stratify cases by observed
HER2 copy number.
Analysis of Results
The HER2 status of all cases measured by immunohistochemical analysis during this period is included. HER2 was
measured by FISH (dual color) in all cases referred for FISH
testing because they had immunohistochemical scores of 2+8,9
and also in a cohort in which all samples were screened by
dual-color FISH. FISH data were analyzed by the number of
cases categorized by the observed mean HER2 copy number
per cell (as in “single-color FISH”). For each HER2 copy
number, the number of cases with HER2 amplification relative
to chromosome 17 (as with dual-color FISH) was determined.
Based on these values, the potential for misdiagnosis of HER2
gene amplification if chromosome 17 was not measured was
calculated. Underdiagnosis was calculated as the proportion of
HER2-amplified cases (by HER2/chromosome 17 ratio) that
would be recorded as HER2– based on the HER2 copy number
if chromosome 17 was not measured. Overdiagnosis was calculated as the number of HER2 nonamplified (by ratio) cases
that would be considered amplified based only on the HER2
copy number if chromosome 17 was not measured.
Results
The HER2 status of 4,903 breast cancer patients was
evaluated by immunohistochemical analysis between 2000
and 2008. Of the 4,903 cases, 63.2% were HER2– (immunohistochemical score, 0 or 1+), 11.4% were scored as 2+, 18.9%
were 3+, and 6.3% had missing immunohistochemical data.
The HER2/chromosome 17 copy ratio was measured by
dual-color FISH in 1,711 of these cases, including all scored
2+ immunohistochemically (593 cases) and also in a cohort in
which all samples were screened by dual-color FISH irrespective of immunohistochemical status. Data on chromosome 17
were missing for 1 case. The patient population with measurement of HER2 by FISH in this study was clearly biased
toward cases scored 2+ immunohistochemically.
The mean HER2 copy number per cell was fewer than
2 in 40.4% of cases, 2 to fewer than 4 in 39.9% of cases, 4
to fewer than 6 in 6.5% of cases, 6 to fewer than 7 in 1.3%
of cases, and 7 or more in 11.8% of cases zFigure 1z. In this
study population of breast cancer patients, the chromosome
17 copy number (recorded from dual-color FISH) was defined
40
Percentage of Cases
35
30
25
20
15
10
5
0
<2
2<3
3<4
4<5
5<6
6<7
7<8
8<9
9<10 10<15 15<20 20<25 >25
HER2 Copy Number
zFigure 1z Distribution of HER2 copy number across the total patient study population. The percentage of cases by observed
mean HER2 copy number per cell is shown. The HER2 copy number was recorded from dual-color fluorescence in situ
hybridization in 1,711 cases.
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© American Society for Clinical Pathology
Anatomic Pathology / Original Article
as monosomic (<1.35) in 9.1% of cases, disomic (1.35-1.85)
in 51.7% of cases, and polysomic (>1.85) in 39.2% of cases
zFigure 2z.
zFigure 3z shows the frequency of HER2 gene amplification (defined by the HER2/chromosome 17 ratio) as a function of observed mean HER2 copy number per cell. zImage
1z illustrates examples of low HER2 copy amplified and high
HER2 copy nonamplified cases. Even at a HER2 copy number
of 2 to fewer than 3, 16 (3.3%) of 488 cases exhibited HER2
amplification; and at a copy number of 3 to fewer than 4, 32
(16.4%) of 195 cases were amplified. The proportion of cases
with HER2 amplification increased considerably at HER2
copy numbers of 4 to fewer than 7: 50.0% at copy numbers
of 4 to fewer than 5; 67.5% at 5 to fewer than 6, and 77.3% at
6 to fewer than 7. Virtually all cases were amplified at HER2
copy numbers of 7 or more.
60
Percentage of Cases
50
40
30
20
10
0
<1.35
1.35-1.85
>1.85-2
>2-3
>3
Chromosome 17 Copy Number
Percentage of Patients With HER2 Amplification
zFigure 2z Distribution of chromosome 17 copy number
across the total patient study population. The percentage
of cases by chromosome 17 copy number per cell is shown.
Chromosome 17 copy number was recorded from dualcolor fluorescence in situ hybridization measurements in
1,710 cases.
Of the 816 cases with a HER2 copy number between 2
and 8, there were 291 cases (35.7%) referred for FISH testing only that, therefore, have no immunohistochemical result
available for analysis; 277 cases (33.9%) with an immunohistochemical score of 2+, reflecting the enrichment of these
cases in this series, of which 27.4% were HER2 amplified;
198 immunohistochemically negative cases (24.3%), of which
only 6 (3.0%) were HER2 amplified; 33 cases (4.0%) with an
immunohistochemical score of 1+, of which 12% were amplified; and 17 cases (2.1%) with an immunohistochemical score
of 3+, of which 82.4% were amplified.
Discussion
Although it is now universally accepted that the vast
majority of patients with early breast cancer require testing
for expression, or preferably amplification, of the HER2
oncogene, debate continues about the appropriate methods to
be used.1-4 A key area of debate is related to when or, indeed,
if chromosome 17 should be evaluated for the determination
of HER2 gene amplification.
HER2 gene amplification is now recognized as the
key driver of HER2 overexpression in breast cancer.6,7
Gene amplification for HER2 has been defined as a ratio of
observed HER2/chromosome 17 copy numbers in invasive
breast cancer cells, which would seem to mandate inclusion
of chromosome 17 for all cases. However, there are clear
hypothetical cases in which exclusion of chromosome 17
may be valid: cases with observed HER2 copy numbers less
than 2.0 are, almost by definition, never amplified, whereas
cases with very high HER2 copy numbers (>20 signals/cell)
are frequently assumed to be amplified. As a result, many
national guidelines have provided estimated HER2 copy number ranges for which testing of chromosome 17 is perceived
100
90
80
70
60
50
40
30
20
10
0
<2
2<3
3<4
4<5
5<6
6<7
7<8
8<9
9<10 10<15 15<20 20<25
>25
HER2 Copy Number (copies per cell)
zFigure 3z Frequency of HER2 gene amplification (defined by HER2/chromosome 17 ratio) as a function of HER2 copy number.
The HER2/chromosome 17 ratio was recorded in 1,711 cases by dual-color fluorescence in situ hybridization.
© American Society for Clinical Pathology
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to be important.1,2,8-11 However, the major weakness of these
guidelines has been the lack of an evidence base for the selection of cases for chromosome 17 testing in centers that use
single-color ISH to assess the HER2 copy number rather than
dual-color ISH for the HER2 ratio. We have addressed this
gap by auditing cases diagnosed in our laboratories by FISH
and using the HER2 gene copy number (derived from dualcolor ISH testing) to estimate the frequency of HER2 gene
amplification (defined by the HER2/chromosome 17 ratio)
relative to HER2 copy number. This analysis suggests that
all guidelines have been excluding a significant proportion
of cases that require chromosome 17 copy number testing to
adequately define HER2 gene amplification.
The potential impact, in terms of misdiagnosis of HER2
gene amplification in this study population, if adhering to current guidelines is illustrated in zTable 1z. Current guidelines
recommend that an observed HER2 copy number of fewer
than 4 should be considered nonamplified1,2 without the need
for chromosome 17 copy number determination. However,
our data show that HER2 is amplified in 16.4% of cases with
an observed mean HER2 copy number of 3 to 4, and 11.4%
of the total patient population tested falls within this group,
which would represent a 1.87% underdiagnosis of HER2 gene
amplification in the total patient study population. In fact,
HER2 is amplified in 3.28% of cases with an observed HER2
copy number of 2 to 3, and 28.5% of the study population falls
A
B
C
D
zImage 1z Low HER2 copy amplified and high HER2 copy nonamplified cases. A (Case 1), Average HER2 copy number, 2.33
per cell. B (Case 1), Average HER2 copy number, 2.33 per cell; average chromosome 17 copy number, 1.00; ratio, 2.33. Case is
amplified according to the ratio. C (Case 2), Average HER2 copy number per cell, 5.05. D (Case 2), Average HER2 copy number
per cell, 5.05; average chromosome 17 copy number, 3.77 per cell; ratio, 1.34. Case is not amplified according to the ratio.
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© American Society for Clinical Pathology
Anatomic Pathology / Original Article
zTable 1z
Potential Percentage of Misdiagnosis in Patient Study Population (N = 1,711) When Adhering to Current Guidelines
Mean HER2 Copy Number
<2
2 to <3
3 to <4
4 to <5
5 to <6
6 to <7
≥7
Percentage of
Total Population
40.44
28.52
11.40
4.21
2.34
1.29
11.80
Percentage of Cases
With HER2 Amplification
Percentage of Misdiagnosis
in Total Population
0.00
3.28
16.41
50.00
67.50
77.27
Virtually 100.00
0.00
0.94
1.87
NA
NA
0.29
NA, current guidelines require chromosome 17 testing in this population, which excludes the potential for misdiagnosis.
within this group, which would represent a further 0.94% of
cases misdiagnosed as HER2 nonamplified when calculated
as a proportion of the total study patient population.
No HER2 amplification was observed in tumors with an
observed HER2 copy number fewer than 2. Therefore, based
on the current guidelines for fewer than 4 HER2 copies per
cell being considered negative,1,2 2.81% of the total patient
population in this study (ie, 1 in 36 patients) would have been
given a misdiagnosis of nonamplified if the chromosome 17
copy number had not been determined. Conversely, a diagnosis of HER2 “amplification” based on a HER2 copy number
of more than 6 to fewer than 7 represents a 22.7% chance of
misdiagnosis because the present study has shown that only
77.3% of these cases have true amplification of HER2 when
the chromosome 17 copy number is measured. Although this
is only 1.29% of the total patient population within this group
(copy number 6 to <7), it still represents a misdiagnosis rate
of 0.3% in the total patient population in this study. Virtually
all cases were amplified at HER2 copy numbers of 7 or more.
Therefore, based on the current guidelines for fewer than 4
HER2 copies per cell being considered negative and 6 or more
HER2 copies per cell being considered positive,1,2 3.1% of
cases in this study population (ie, 1 in 32 patients) would have
received an incorrect diagnosis with respect to HER2 gene
amplification (Table 1).
Current guidelines recommend that a HER2 copy number
of 4 to 6 should be validated by measurement of chromosome
17.1,2 HER2 is amplified in 50.0% of cases with a HER2 copy
number of 4 to fewer than 5, and 4.2% of the study population
falls within this group (Table 1). Similarly, HER2 is amplified
in 67.5% of cases with a HER2 copy number of 5 to fewer than
6, and 2.34% of the study population falls within this group. If
all cases with a copy number of 4 to 6 were considered positive, as is the case in some centers that ignore the importance
of measuring chromosome 17, this would represent a further
significant population of cases with an incorrect diagnosis with
respect to HER2 gene amplification (2.9%).
For centers using single-probe ISH (FISH or CISH)
for the detection of amplification of the HER2 gene, current
© American Society for Clinical Pathology
guidelines mandate testing for chromosome 17 copy number
for cases with an observed HER2 gene copy number between
4 and 6 copies per cell.1,2 An audit of almost 1,700 cases, for
which FISH was performed in our laboratories, demonstrates
that these guidelines are important and prevent the misdiagnosis of a significant group of HER2-amplified breast cancers
in laboratories using single-color ISH assays (2.9% of cases;
ie, 1/34 of all cases). However, we also showed that an equal
proportion of cases (3.1%; 1/32) are at risk of misdiagnosis
even in laboratories adhering to current guidelines for chromosome 17 testing following single-color ISH for HER2.
Extending the use of chromosome 17 analysis to all cases with
observed HER2 copy numbers between 2.0 and 7.0 per cell
would avoid the current misdiagnosis of HER2 gene amplification for these cases. Even in this population with low copy
numbers, there seems to be a strong relationship between gene
amplification and HER2 protein expression. However, these
data are compromised by the large proportion of cases scored
immunohistochemically as 2+ and cases for which no immunohistochemical result was available for analysis.
We, therefore, recommend that all cases with observed
HER2 copy numbers of 2 to 7 should also be analyzed
for chromosome 17 to accurately determine HER2 gene
amplification. zTable 2z illustrates the impact of this recommendation, which would require analysis of chromosome
17 in 48.3% of all breast cancer cases based on the study
population. Current guidelines1,2 recommend chromosome
17 measurement only in cases with a HER2 copy number of
4 to fewer than 6, which represents only 6.6% of all breast
cancer cases in the present study. Although single-color ISH
is becoming more widely used with the availability of CISH,
the importance of chromosome 17 measurement cannot be
ignored. It is essential that HER2 testing is of high quality, so
that optimal patient management can be provided. Because
HER2+ status correlates with clinical efficacy of trastuzumab,
it is necessary to accurately identify patients who will benefit
from this treatment and to avoid unnecessary treatment of
patients who are unlikely to benefit. False-positive HER2
results can give rise to overtreatment of patients, raise false
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zTable 2z
Percentage of Total Patient Study Population (N = 1,711)
Requiring Chromosome 17 Measurement if Chromosome 17
Was Measured in All Cases
HER2 Copy Number
4 to <6
3 to <6
2 to <6
2 to <7
Percentage of Total Population Requiring
Chromosome 17 Measurement
6.6
18.2
47.0
48.3
hope, and waste resources, whereas false-negative results may
result in undertreatment and deny women with breast cancer
potentially life-extending treatment. The issue of the potential
for misdiagnosis and requirement for greater accuracy has
recently been highlighted in the press.20
We recommend that all breast cancer cases with a HER2
copy number of 2 to 7 determined by single-color ISH also
be analyzed for chromosome 17 to obtain a more accurate
diagnosis of HER2 amplification.
From the 1Endocrine Cancer Group, Edinburgh University
Cancer Research Centre, Western General Hospital, Edinburgh,
Scotland; and 2Department of Pathology, Western Infirmary,
Glasgow, Scotland.
Diagnostic HER2 testing supported in part by Roche
Products, Welwyn Garden City, England. J. Merritt, PhD, Merritt
Science, St Albans, England, a professional medical writer,
drafted the manuscript and was paid by Roche Products.
Address reprint requests to Dr Bartlett: Endocrine Cancer
Group, Edinburgh University Cancer Research Centre, Western
General Hospital, Crewe Rd South, Edinburgh EH4 2XR, Scotland.
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© American Society for Clinical Pathology