Anatomic Pathology / HER2/NEU AMPLIFICATION IN BREAST CANCER
HER2/neu Amplification in Breast Cancer
Stratification by Tumor Type and Grade
Elise R. Hoff, MD, Raymond R. Tubbs, DO, Jonathan L. Myles, MD, and Gary W. Procop, MD
Key Words: Breast cancer; HER2/neu; c-erbB-2
Abstract
The presence of HER2/neu gene amplification is
prognostically and therapeutically significant for
patients with breast cancer. We sought to determine
whether a relationship exists between HER2/neu gene
amplification and the histologic type and grade of
tumor. The histologic features and corresponding
HER2/neu amplification results of 401 cases of invasive
breast carcinoma were reviewed. Lobular carcinomas
were less likely than ductal carcinomas to have
HER2/neu amplification. Amplification was less
frequent in Scarff-Bloom-Richardson grade 1 ductal
carcinomas than in grades 2 and 3. Metastatic
carcinomas frequently displayed HER2/neu
amplification (6/20 [30%]). Our results support a
correlation between HER2/neu amplification and the
histologic type and grade of breast cancer. We suggest
reexamination of tumors diagnosed as Scarff-Bloom-
Richardson grade 1 invasive ductal carcinomas or
lobular carcinomas if the lesion displays HER2/neu
amplification to assure the exclusion of a higher grade
of lesion or of missed ductal components.
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The annual incidence of breast carcinoma in the United
States in 2000 was estimated at 182,000 cases, and the annual
number of deaths was estimated at 41,000 persons.1 Although
mortality rates declined during the mid-1990s, breast cancer
remains a leading cause of cancer death among women,
second only to lung cancer. Morphologic variables, such as
tumor size, grade, and metastases to regional lymph nodes, are
important prognostic indicators for patients with breast cancer.
In addition, amplification of the HER2/neu proto-oncogene,
which has been reported to occur in 10% to 34% of invasive
breast carcinomas, also has been shown to be of both prog-
nostic and therapeutic significance.2,3 This molecular variable
may be used to guide therapy and to stratify patients into clini-
cally relevant risk groups. Detection of the amplification of
the HER2/neu oncogene or the expression of the protein
product it encodes is now performed widely in the United
States. HER2/neu testing is performed routinely on all inva-
sive breast carcinomas at the Cleveland Clinic Foundation,
Cleveland, OH. We sought to determine whether a relation-
ship exists between the presence of HER2/neu amplification
and the type of breast cancer (ie, invasive ductal carcinoma or
invasive lobular carcinoma) and the Scarff-Bloom-Richardson
(SBR) grade for invasive ductal carcinomas.
Materials and Methods
Clinical Samples
The files of the Cleveland Clinic Foundation were searched
for all cases of invasive breast carcinoma that were diagnosed
between July 1999 and July 2000, on which fluorescent in situ
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hybridization was performed for the HER2/neu gene. We
identified 401 cases. Of these, 388 were diagnosed as inva-
sive ductal, invasive lobular, or metastatic breast carcinoma.
The remaining 13 consisted of special-type tumors, such
as tubular, medullary, inflammatory, secretory, and colloid
carcinomas; HER2/neu results on these tumors were
recorded, but statistical analysis was not performed because
of the low number of each tumor type. Histologic assessment
of tumor type and modified SBR grading were routinely
performed on 5-m-thick, H&E-stained sections of the
formalin-fixed, paraffin-embedded tumors by the attending
pathologist. For this study, all tumors were assigned to 1 of 3
groups: invasive ductal carcinoma, invasive lobular carci-
noma, or metastatic breast cancer. Invasive ductal carci-
nomas were further separated into 3 subgroups based on
SBR grade (SBR grade 1, SBR grade 2, or SBR grade 3).
The metastatic carcinomas were analyzed together as a
group, regardless of type or grade of the primary lesion,
which was not always known. Cases designated as invasive
lobular carcinoma included tumors that demonstrated
complete lack of duct formation and had typical lobular
features; pleomorphic lobular carcinomas also were included
in the invasive lobular carcinoma subgroup.
In Situ Hybridization
Unstained sections on electrostatically charged slides
were heated for 30 minutes at 60C, then deparaffinized in 2
changes of xylene (5 minutes each), followed by 2 changes
of absolute ethanol (1 minute each). The sections then under-
went cell conditioning in a 95C water bath, immersed in a
target-retrieval solution (DAKO, Carpinteria, CA) for 40
minutes. After cooling at room temperature for 20 minutes,
they were rinsed in distilled water and digested with
Proteinase K (150 L diluted 1:5,000 in 50 mmol of
tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6,
DAKO). Enzymatic action was stopped with distilled water
rinses. After dehydration in graded alcohol, digoxigenin-
labeled HER2/neu probe (10 L, Ventana, Tucson, AZ) was
applied to the sections, which then were heated at 90C for 6
minutes to allow for denaturing of DNA. Hybridization of
probe and target tissue DNA took place overnight in a 37C
incubator. Stringency washes of 0.5 standard saline citrate
for 5 minutes at 72C followed. The slides then were washed
in 1 phosphate-buffered saline containing 0.1% polysor-
bate-20 for 5 minutes, after which fluorescein-labeled
antidigoxigenin antibody was applied. The slides then were
counterstained with 20 L of 4',6-diamidino-2-phenylindole
in antifade solution. Signals were visualized on an Axioskop
(Zeiss, Oberkochen, Germany). Gene copies were counted in
2 preselected fields, 20 nuclei in each field, for a total of 40
nuclei. Amplification was recorded as absent (1-4 gene
copies) or amplified (>4 copies).
American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
Statistical Methods
The following parameters were determined for each of
the histologic subgroups: frequency of HER2/neu gene
amplification, mean number of gene copies, and range of
gene copy number for cases with HER2/neu amplification.
To further stratify the data, the number of cases in each cate-
gory was separated into cases with 5 to 10 signals per
nucleus and those with more than 10 signals per nucleus. A
comparison of the frequency of HER2/neu amplification
with the type and grade of carcinoma was performed, using
the chi-square test with Yates correction.
Results
A total of 388 cases of invasive ductal carcinoma, inva-
sive lobular carcinoma, and metastatic breast cancer were
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identified. Of these, 300 were invasive ductal carcinomas
(SBR grade 1, 73; SBR grade 2, 106; SBR grade 3, 121), 68
were invasive lobular carcinomas, and 20 were metastatic
tumors (3 to axillary lymph nodes, 8 to the chest wall, 4 to
bone, 2 to lung, and 3 to the liver).
Only 1 (<1%) of 73 SBR grade 1 invasive ductal carci-
nomas demonstrated HER2/neu amplification compared
with 17.0% (18/106) of the SBR grade 2 and 23.1% (28/121)
of the SBR grade 3 invasive ductal carcinomas Image 1,
Image 2, Image 3, and Image 4. Only 2 (3%) of 68 inva-
sive lobular carcinomas demonstrated HER2/neu amplifica-
tion. The metastatic carcinomas, although limited in number
in this review (n = 20), demonstrated the highest frequency
of amplification: 30% (6/20). None of the 13 special-type
tumors, which consisted of tubular, medullary, inflammatory,
secretory, and colloid carcinomas, demonstrated HER2/neu
gene amplification.
Comparison of these frequencies using the chi-square
test with Yates correction revealed statistically significant
differences in the frequencies of amplification between inva-
sive lobular (2/68) and invasive ductal carcinomas (47/300)
(P < .005). Significant differences also were found between
the frequency of amplification in SBR grade 1 invasive
ductal carcinomas compared with SBR grade 2 and grade 3
invasive ductal carcinomas (P < .005 and P < .001, respec-
tively). There was no significant difference in HER2/neu
amplification between SBR grade 2 and SBR grade 3 inva-
sive ductal carcinomas. Of 18 SBR grade 2 invasive ductal
carcinomas that demonstrated HER2/neu amplification, 6
(33%) had 5 to 10 copies per nucleus, while 12 (67%) had
more than 10 copies per nucleus. Of 28 SBR grade 3 inva-
sive ductal carcinomas that demonstrated HER2/neu amplifi-
cation, 13 (46%) had 5 to 10 copies per nucleus, while 15
(54%) had more than 10 copies per nucleus. Of 6 metastatic
carcinomas, 4 (67%) had more than 10 copies per nucleus,
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Hoff et al / HER2/NEU AMPLIFICATION IN BREAST CANCER
Image 1 Typical low-grade (Scarff-Bloom-Richardson grade 1)
invasive ductal carcinoma demonstrating prominent tubule
formation, low nuclear grade, and few to absent mitoses
(H&E, 400).
Image 3 Typical high-grade (Scarff-Bloom-Richardson grade
3) invasive ductal carcinoma demonstrating lack of tubule
formation, high nuclear grade, and prominent mitoses (H&E,
400).
while the remaining 2 (33%) had only 5 to 10 copies per
nucleus. The single SBR grade 1 invasive ductal carcinoma
that demonstrated HER2/neu amplification had 19.4 copies
per nucleus. The 2 lobular carcinomas that had amplification
of the HER2/neu gene both had 5 to 10 copies per nucleus.
The mean and range of HER2/neu copy numbers for cases
with gene amplification are given in Table 1.
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Image 2 Lack of amplification of the HER2/neu gene was
seen typically in low-grade invasive ductal carcinomas (2
gene copies per nucleus) (1,000).
Image 4 Fluorescence in situ hybridization image of a high-grade
(Scarff-Bloom-Richardson grade 3) carcinoma showing presence
of amplification of the HER2/neu gene. The large green signals
represent the blurring of signals that occurs when large numbers
of copies are present in proximity to each other (400).
Because only 2 of 68 invasive lobular carcinomas
demonstrated HER2/neu amplification, the lobular carci-
nomas were reexamined to exclude the possibility of a
misclassification. One of these cases was a pleomorphic
variant of lobular carcinoma, with a nuclear grade of 3/3.
The other case was reexamined by 3 pathologists with exper-
tise in breast pathology and determined to represent not an
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Table 1
HER2/neu Amplification Stratified by Tumor Type and Grade
No. (%) of Cases
Tumor Type No. of Cases With Amplification
Ductal (SBR grade)
1 73 1 (1)
2 106 18 (17.0)
3 121 28 (23.1)
Lobular 68 2 (3)*
Metastatic 20 6 (30)
Total 388 55 (14.2)
SBR, Scarff-Bloom-Richardson.
* Reexamination of the histopathologic features of the 2 cases of lobular carcinoma that demonstrated HER2/neu amplification revealed one to be a pleomorphic variant of
lobular (nuclear grade 3/3), while the other had been misclassified; it was determined to be an invasive ductal carcinoma, SBR grade 2. Therefore, 1/67 (1%) of invasive lobular
carcinomas demonstrated HER2/neu amplification. A recalculation because of the reclassified case was not performed, since significant differences already had been
established.
invasive lobular carcinoma but rather an invasive ductal
carcinoma (SBR grade 2); rare ductal structures were identi-
fied. Based on this reassessment, we believe only 1 (<1%) of
the now 67 cases of invasive lobular carcinoma, a pleomor-
phic variant of lobular carcinoma, actually had amplification
of the HER2/neu oncogene. In a similar manner, the 1 SBR
grade 1 invasive ductal carcinoma that demonstrated
HER2/neu amplification was reassessed to ensure the exclu-
sion of a higher-grade lesion. In this case, however, all 3
pathologists agreed with the original diagnosis.
Discussion
HER2/neu is a proto-oncogene located on the long arm
of chromosome 17.4 Many adult tissues, including breast,
endometrium, prostate, and ovary, normally express low
levels of the protein encoded for by this gene. Amplified
levels of this gene and its protein product have been found in
between 10% and 35% of invasive breast carcinomas. 2
HER2/neu amplification in breast cancer has been associated
with a number of adverse outcomes, including decreased
overall and disease-free survival, especially for patients with
disease metastatic to lymph nodes, 5-7 Because of its
numerous adverse associations, HER2/neu amplification
status has become an increasingly important and reliable
predictor of patient outcome, and testing for this variable is
now widely performed. Determination of this variable also
has become an important aid in the determination of which
patients will be candidates for the new anti-HER2/neu drug,
trastuzumab (Herceptin), which has been reported to be of
benefit to patients with breast cancers that overexpress
HER2/neu.8-10 At our institution, all cases of invasive breast
carcinoma are tested for HER2/neu amplification. In this
retrospective review, we sought to ascertain whether signifi-
cant differences exist between the type of breast cancer, the
American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
No. of Cases No. of Cases With Mean (Range) of Gene
With 5-10 Copies >10 Copies Copy Nos. in Cases
per Nucleus per Nucleus With Amplification
0 1
6 12 14 (6 to 20.0)
13 15 11.6 (5.1 to >20.0)
2 0 6.4 (6.2 to 6.7)
2 4 12.7 (5.2 to 19.0)
23 32
grade of invasive ductal carcinomas, and the HER2/neu
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amplification status.
We found that overall, invasive ductal carcinomas were
significantly more likely to show HER2/neu amplification
than were invasive lobular carcinomas (P < .005). In addi-
tion, higher grade invasive ductal carcinomas (SBR grades 2
and 3) were more likely to demonstrate HER2/neu amplifica-
tion (17.0% and 23.1%, respectively) than lower grade (SBR
grade 1) ductal carcinomas (1%). These differences were
statistically significant (P < .005 and P < .001, respectively).
The fact that most of the invasive lobular carcinomas
(66/67 [99%]) and low-grade (SBR grade 1) invasive ductal
carcinomas (72/73 [99%]) in our study lacked HER2/neu
amplification suggests that amplification is highly unlikely in
these types of carcinomas. The presence of HER2/neu ampli-
fication in pleomorphic lobular carcinoma was less
surprising, given its high nuclear grade. In this study, we
identified a tumor that demonstrated HER2/neu amplifica-
tion; the tumor originally was suspected to be a lobular carci-
noma, but on further study was found to be an invasive
ductal carcinoma, SBR grade 2. Therefore, the presence of
HER2/neu amplification in an invasive lobular carcinoma or
an SBR grade 1 invasive ductal carcinoma should prompt
reevaluation of the tumor to exclude the possibility of
misclassification.
This low frequency of amplification among the lobular
carcinomas in our study correlates with results found by
Porter et al,11 who examined c-erbB-2 expression in cases
containing in situ and invasive lobular carcinomas. c-erbB-2
expression was found in none of their 15 cases containing
invasive lobular carcinoma, and it was present in only 1 of
the 57 cases with in situ lobular carcinoma. This case was
described as having only weak staining, which may not have
represented true amplification.11 Similarly, Rosenthal et al12
also found that invasive lobular carcinomas were much less
likely than invasive ductal carcinomas to demonstrate
Am J Clin Pathol 2002;117:916-921 919
Hoff et al / HER2/NEU AMPLIFICATION IN BREAST CANCER
HER2/neu amplification (13% compared with 48%). The
lobular carcinomas they tested, however, demonstrated a
much higher frequency of amplification than the lobular
carcinomas in the present review: 13% compared with 1%
(1/68). Rosenthal et al12 also found HER2/neu amplifica-
tion to be as significant an adverse prognostic factor among
the lobular carcinomas as it was among the ductal carci-
nomas. Other investigators also have reported similar
differences in rates of HER2/neu amplification between
ductal and lobular carcinomas.13-16 In contrast, however,
Rosen et al,17 in a study of HER2/neu expression and tumor
phenotype, reported HER2/neu amplification in ductal and
lobular carcinomas, and they found almost equal rates of
amplification in these groups: 49% in ductal carcinomas
and 43% in lobular carcinomas. These authors did not find
a relationship between the grade of tumor differentiation
and HER2/neu expression. The reason for the higher
percentage of amplified lobular carcinomas in the study by
Rosen et al17 compared with ours may be related to the
different methods used (immunohistochemical analysis vs
fluorescent in situ hybridization) and to the criteria they
used to define positivity: 25% or more of carcinoma cells
showing membrane immunoreactivity. Some authors may
consider this cutoff value as too low to qualify for overex-
pression of HER2/neu.18 Therefore, the higher percentage
of amplified lobular carcinomas found by Rosen et al17
may have been due to overinterpretation of the immunohis-
tochemical staining, resulting in the inclusion of possible
false-positive results.
Other studies have compared HER2/neu expression
with the tumor grade of invasive ductal carcinomas.14,15,17,19-22
In a 1991 study by Rilke et al,21 consisting of 1,210 patients,
amplification was found in 3.9% of grade 1 carcinomas,
20.4% of grade 2 carcinomas, and 38.9% of grade 3 carci-
nomas. Similarly, Tsuda et al19 found c-erbB-2 amplifica-
tion in 33% of grade 3 invasive ductal carcinomas, 10% of
grade 2 carcinomas, and 0% of grade 1 carcinomas. The
values from these studies are similar to those found in the
present study.
These data support the existence of a correlation
between HER2/neu amplification and both tumor type and
histologic grade of the invasive ductal carcinoma. Only 1
of our grade 1 ductal carcinomas and 1 of the lobular
carcinomas, a pleomorphic variant, demonstrated
HER2/neu amplification; none of the nonpleomorphic
variants of lobular carcinoma demonstrated HER2/neu
amplification. Interestingly, one of the carcinomas that was
diagnosed originally as an infiltrating lobular carcinoma but
displayed HER2/neu amplification was found to be an inva-
sive ductal carcinoma on review. Therefore, we suggest that
if HER2/neu amplification is present in SBR grade 1 inva-
sive ductal carcinomas or in invasive lobular carcinomas,
920 Am J Clin Pathol 2002;117:916-921
reexamination of the morphologic features of the neoplasm
should be performed to confirm the tumor type and grade
as a matter of quality assurance.
From the Departments of Anatomic and Clinical Pathology,
Cleveland Clinic Foundation, Cleveland, OH.
Address reprint requests to Dr Procop: Dept of Pathology,
MailStop L40, Cleveland Clinic Foundation, 9500 Euclid Ave,
Cleveland, OH 44195.
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