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Anatomic Pathology / HER-2/NEU AMPLIFICATION AND HER-2/NEU OVEREXPRESSION
HER-2/neu Gene Amplification Compared With HER-2/neu
Protein Overexpression and Interobserver Reproducibility
in Invasive Breast Carcinoma
Mai P. Hoang, MD, Aysegul A. Sahin, MD, Nelson G. Ordòñez, MD, and Nour Sneige, MD
Key Words: HER-2/neu; c-erbB-2; Fluorescence in situ hybridization; Gene amplification; Herceptin; Breast carcinoma;
Immunohistochemistry; HercepTest; Interobserver reproducibility
Abstract
We compared the detection of HER-2/neu gene
amplification by fluorescence in situ hybridization
(FISH) with detection of HER-2/neu protein
overexpression by immunohistochemistry using 2
antibodies on 100 archival invasive breast carcinomas.
Protein overexpression for each marker was scored
independently by 4 pathologists using standardized
criteria, and consensus was compared with results
obtained from gene amplification. The concordance rate
between FISH and immunohistochemistry was 76% for
e2-4001 and 91% for the HercepTest. Of the 37 cases
positive by e2-4001, 21 demonstrated no gene
amplification; 7 of 24 cases positive by the HercepTest
demonstrated no gene amplification. However, 1 of 61
cases negative by e2-4001 showed gene amplification;
none of the cases negative by the HercepTest showed
amplification. The predictive values of gene
amplification based on 0-1+, 2+, and 3+
immunohistochemical staining were best for cases
scored as 3+ (75% for e2-4001 and 89% for the
HercepTest). Complete agreement among observers for
immunohistochemical scoring of e2-4001 and the
HercepTest was achieved in 75 and 85 cases,
respectively. The pairwise kappa agreement values were
substantial for e2-4001 and substantial to almost perfect
for the HercepTest. Immunohistochemical staining may
be considered a useful screening test. While negative
staining almost always correlated with a lack of gene
amplification, positive membranous staining, especially
2+, did not predict gene amplification. The low
interobserver reproducibility in separating 2+ from 3+
cases necessitates further confirmation by FISH before
treatment decisions are made.
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Carcinoma of the breast is the most common malignant
neoplasm in women in the United States, and only lung
cancer causes more cancer deaths in women.1 The HER2/neu proto-oncogene is amplified and overexpressed in 25%
of invasive breast carcinomas, and this alteration predicts
shortened disease-free survival and poor clinical outcome in
cases of breast carcinoma.2-5 Also known as c-erbB-2, the
HER-2/neu (human epidermal growth factor receptor) gene is
located at 17q12-21.32 and is thought to be involved in the
control of cell growth and development.6 It encodes a transmembrane 185-kd tyrosine kinase growth factor receptor
similar to but distinct from the epidermal growth factor
receptor.7 Amplification of the HER-2/neu proto-oncogene
leads to marked overexpression of the membrane-bound
growth factor receptor protein at the cell surface.8 Given the
location of the receptor at the cell’s external surface, there has
been considerable interest in developing and using specific
neutralizing monoclonal antibodies that block the signaling
function of these receptor tyrosine kinases. Recently, the use
of recombinant humanized anti-p185HER-2/neu monoclonal
antibody (Herceptin, Genentech, San Francisco, CA) in
combination with cisplatin has been shown to result in clinical response in patients whose metastatic breast cancer is
refractory to chemotherapy treatment and overexpresses
HER-2/neu.9
The standard method to analyze HER-2/neu gene amplification has been the Southern blot hybridization test.
However, this procedure requires nondegraded DNA,
meaning that a large specimen, either fresh or frozen, is
needed. Dilutional artifacts from the inclusion of normal cells
also can mar the procedure’s results. Because it often is
necessary to analyze small, paraffin-embedded tissues, other
methods, such as fluorescence in situ hybridization (FISH)
© American Society of Clinical Pathologists
Anatomic Pathology / ORIGINAL ARTICLE
and immunohistochemistry have been developed. By using
specific proto-oncogene sequences as probes, FISH enables
the detection of the amplified copy number on a cell-by-cell
basis. On the other hand, immunohistochemistry measures
the HER-2/neu protein overexpression at the membrane of
the tumor cells.
Although FISH and immunohistochemical (DAKO
HercepTest, DAKO, Carpinteria, CA) methods have been
approved by the US Food and Drug Administration as clinical tests for breast carcinoma,10 the question remains as to
which is the better method to assess the HER-2/neu status of
this disease, especially in the context of Herceptin therapy.11
Comparative studies of the application of FISH vs immunohistochemistry on paraffin-embedded breast carcinomas
were published recently.12-14 We evaluated the detection of
HER-2/neu gene amplification by FISH in comparison with
the detection of HER-2/neu protein overexpression by 2 antibodies (one present in the DAKO HercepTest; the other, the
Neomarkers monoclonal antibody e2-4001, Labvision,
Union City, CA) on archival tissues of 100 invasive breast
carcinomas. While the HercepTest is the only immunohistochemical test approved by the US Food and Drug Administration for this purpose, the Neomarkers monoclonal e2-4001
has been used by our laboratory for years, and changing to
the HercepTest would result in a major increase in costs to
the patients. For this reason, we included e2-4001 in our
study. We also assessed interobserver variability in the evaluation of these 2 antibodies.
Materials and Methods
Case Selection
The study material consisted of specimens from 100
consecutive cases of invasive breast carcinoma diagnosed at
or referred to The University of Texas M.D. Anderson
Cancer Center, Houston, in which archival material was
available and the FISH test had been requested. The corresponding H&E-stained slides from all cases were reviewed.
Unstained tissue sections containing tumor, as well as adjacent normal breast parenchyma when possible and corresponding to those used for the FISH test, were selected from
each case for immunohistochemical staining.
Fluorescence In Situ Hybridization
FISH analyses were performed using the PathVysion
HER-2 DNA Probe Kit (Vysis, Downers Grove, IL) according
to the manufacturers’ instructions and using reagents, probes,
and positive controls purchased from the manufacturers. The
slides were evaluated for HER-2/neu gene copy number using
an epifluorescence microscope (Zeiss, Thornwood, NY).
© American Society of Clinical Pathologists
The PathVysion kit uses 2 directly labeled fluorescent
DNA probes: LSI HER-2/neu, which is specific for the
HER-2/neu gene locus, and CEP 17, which is specific for the
alpha satellite DNA sequence at the centromeric region of
chromosome 17. The expected ratio of LSI HER-2/neu to
CEP 17 is 2.0 for normal or unamplified breast tissue specimens. A ratio of greater than 2.0 was considered amplified.
Signals were counted for 60 tumor nuclei within an area of
invasive carcinoma.
Signal enumeration was performed following the criteria
established by Hopman et al15: overlapping nuclei were not
counted, and split signals were counted as 1 chromosome
component. The ductal epithelial cells served as the internal
control. Stromal and inflammatory cells were excluded from
analysis on the basis of the morphologic features of their
nuclei.
Immunohistochemical Studies
For immunohistochemical studies, 4-µm-thick sections
were cut from the same paraffin block that was used for the
FISH analysis, mounted on charged slides, deparaffinized
in xylene, and rehydrated in descending grades (100%70%) of ethanol. Sections were then subjected to heatinduced epitope retrieval by immersion in a 0.01-mol/L
concentration of citrate buffer (pH 6.0) preheated to more
than 90°C and heated in an electric vegetable steamer
(Black and Decker, Shelton, CT) for 54 minutes. Endogenous peroxidase activity was blocked by a 5-minute treatment with 3% hydrogen peroxide in absolute methanol.
The slides were incubated with the anti–HER-2/neu e24001 monoclonal antibody (1:100 dilution) on an
autostainer (DAKO) for 60 minutes at room temperature.
The immunoperoxidase staining was done using the
LSAB2 peroxidase kit (DAKO). The antigen-antibody
immunoreaction was visualized using 3-3´-diaminobenzedine as the chromogen. The slides were counterstained with
Mayer hematoxylin. Similar sections also were stained for
HER-2/neu according to the instructions of the manufacturer and using the reagents included in the HercepTest kit.
The primary antibody included in the kit was polyclonal,
raised in rabbits, and provided in a prediluted form. For
each run, a composite slide of 3 formalin-fixed human
breast carcinoma cell lines representing different levels of
HER-2/neu protein expression (MDA-231 [0], MDA-175
[1+], and SKBR3 [3+]) was used as the control. In addition, for each case, 1 slide was incubated with normal
rabbit serum instead of the primary antibody and used as a
negative control.
The immunohistochemical preparations were interpreted
without knowledge of the results of the FISH tests and
following the criteria recommended by DAKO for the
HercepTest. Overexpression of HER-2/neu was defined as
Am J Clin Pathol 2000;113:852-859
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Hoang et al / HER-2/NEU AMPLIFICATION AND HER-2/NEU OVEREXPRESSION
positive membranous staining in more than 10% of the
neoplastic cells. Partial or incomplete, weak to moderate, and
moderate to strong membranous staining in more than 10%
of the tumor cells were scored as 1+ (negative), 2+ (weak
positive), and 3+ (strong positive), respectively. Borderline
cases between 1+ and 2+ were considered to be equivocal or
indeterminate. The intensity of the staining of the adjacent
normal ductal epithelium also was scored.
Seventy-eight tumors were primary, and 22 represented metastases (to the lymph nodes in 18 cases, the liver in 2 cases, and
to the stomach and to bone marrow in 1 case each).
Fluorescence In Situ Hybridization
FISH analysis was performed successfully on all 100
cases. There was 1 single fluorescent signal for each copy of
the HER-2/neu gene. Seventeen (17%) cases showed gene
amplification. The HER-2/neu–CEP17 ratio ranged from 2.3
to 13.2 (median, 11.8; mean, 8.6).
Interobserver Reproducibility
Four pathologists (M.P.H., A.A.S., N.G.O., N.S.) independently evaluated the immunohistochemical preparations
in a test of interobserver reproducibility. Discrepant cases
were reviewed at a multiheaded microscope to achieve
consensus. The level of interobserver agreement was quantitated using the generalized kappa and pairwise kappa statistics.16 The pairwise kappa statistic is the proportion of cases
in which 2 observers agree, adjusted for the level of agreement that would be expected to occur solely by chance. The
generalized kappa is the summary of the agreement across
all observers. The consensus opinion was viewed as conclusive. Interpretations for kappa are as described by Landis and
Koch ❚Table 1❚.17
Immunohistochemical Studies and Interobserver
Reproducibility
e2-4001
HER-2/neu protein overexpression (consensus score)
was detected in 37 (37%) of 100 analyzed cases. Sixty-one
(61%) cases demonstrated no protein overexpression, and 2
(2%) cases were equivocal ❚Table 2❚.
Complete agreement among the 4 observers was
achieved in 75 cases (75%). Generalized kappa values indicated a substantial agreement (0.77; 95% confidence
interval). The pairwise kappa value ❚Table 3❚ for agreement
among observers was substantial (0.71 to 0.80). ❚Table 4❚
shows the proportion of cases assigned to each category of
immunohistochemical score stratified by observers. There
were significant differences in the way the observers scored
cases in the weakly positive category (9%-18%). However,
the differences in scoring were reduced when the weak and
strong positive cases were combined. Good agreement was
seen in the negative immunohistochemical category, with
assigned percentages ranging from 61% to 64%.
The overall concordance rate between gene amplification
and protein overexpression was 75% (P < .0001). Correlation
between immunohistochemical score and FISH is shown in
❚Table 5❚. Gene amplification was detected in 1 of the 61
cases with negative (0-1+) immunohistochemical staining, in
neither of the 2 equivocal cases, in 1 of the 17 cases with
weak (2+) immunohistochemical staining, and in 15 of the 20
cases with strong (3+) immunohistochemical staining (Table
5). As a result, the prediction of gene amplification based on
Results
Clinicopathologic Data
The age of the patients at diagnosis ranged from 23 to 78
years (median, 51 years). Disease stage varied from T1 to T4.
❚Table 1❚
Interpretations for kappa Scores
kappa Statistic
Strength of Agreement
<0.00
0.00-0.20
0.21-0.40
0.41-0.60
0.61-0.80
0.81-1.00
Poor
Slight
Fair
Moderate
Substantial
Almost perfect
❚Table 2❚
Fluorescence In Situ Hybridization (FISH) vs Immunohistochemical Results by Consensus Score for 100 Cases*
Immunohistochemical Results
e2-4001
Overexpression
FISH
Amplification
No amplification
Total
16 (16)
21 (21)
37 (37)
No Expression
1 (1)
60 (60)
61 (61)
HercepTest
Equivocal
0 (0)
2 (2)
2 (2)
Overexpression
17 (17)
7 (7)
24 (24)
No Expression
0 (0)
74 (74)
74 (74)
Equivocal
0 (0)
2 (2)
2 (2)
* Data are given as number (percentage). Neomarkers monoclonal antibody e2-4001, Labvision, Union City, CA; HercepTest, DAKO, Carpinteria, CA.
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© American Society of Clinical Pathologists
Anatomic Pathology / ORIGINAL ARTICLE
❚Table 3❚
Pairwise kappa Values Among Observers and With Consensus Score*
Observer
e2-4001/Observer
1
2
3
4
HercepTest/Observer
1
2
3
4
2
3
4
Consensus
0.79
0.75
0.77
0.80
0.80
0.71
0.91
0.82
0.78
0.88
0.88
0.76
0.72
0.83
0.76
0.72
0.88
0.90
0.77
0.83
* Neomarkers monoclonal antibody e2-4001, Labvision, Union City, CA; HercepTest, DAKO, Carpinteria, CA.
❚Table 4❚
Percentages of Cases Assigned to Each Category of Immunohistochemical Intensity by Each Observer*
Test/Observer
e2-4001
1
2
3
4
Consensus
HercepTest
1
2
3
4
Consensus
Negative
Equivocal
Weak Positive
Strong Positive
Weak and Strong Positive
64
62
63
61
61
0
0
0
4
2
18
15
9
13
21
18
23
28
22
16
36
38
37
35
37
75
73
72
74
74
0
1
5
0
2
7
6
3
10
7
18
20
20
16
17
25
26
23
26
24
* Neomarkers monoclonal antibody e2-4001, Labvision, Union City, CA; HercepTest, DAKO, Carpinteria, CA.
❚Table 5❚
Predictive Value of Immunohistochemical Results and
Correlation Between Intensity and Fluorescence In Situ
Hybridization (FISH)*
Immunohistochemical Results
e2-4001
Negative, 0-1+ (n = 61)
Equivocal (n = 2)
Weak positive, 2+ (n = 17)
Strong positive, 3+ (n = 20)
HercepTest
Negative, 0-1+ (n = 74)
Equivocal (n = 2)
Weak positive, 2+ (n = 6)
Strong positive, 3+ (n = 18)
No. (%) of Cases
Amplified by FISH
1 (2)
0 (0)
1 (6%)
15 (75)
0 (0)
0 (0)
1 (17)
16 (89)
* Neomarkers monoclonal antibody e2-4001, Labvision, Union City, CA;
HercepTest, DAKO, Carpinteria, CA.
immunohistochemical results was 75% for those with strong
positive (3+) immunohistochemical staining, 6% for the
weak positive (2+) cases, and 2% for the cases negative by
immunohistochemical studies. Benign ductal epithelium was
present in 54 cases. Three of the 20 cases with 2+ and 3+
© American Society of Clinical Pathologists
positivity demonstrated membranous staining. If this finding
is considered,12 the immunohistochemical results for such
cases would be considered negative; however, the predictive
value for the remaining 17 cases is still low (7%). Of note,
the percentage of positive tumor cells varied from 20% to
100% among positive cases, while it was more than 80% in
15 of 16 cases with concordant FISH and immunohistochemical results.
HercepTest
HER-2/neu protein overexpression (consensus score)
was detected in 24 (24%) of 100 cases analyzed. Seventyfour cases (74%) demonstrated no protein overexpression,
and 2 cases (2%) were equivocal (Table 2).
Complete agreement among the 4 observers was
achieved in 85 cases (85%). Generalized kappa values indicated a substantial agreement (0.78; 95% confidence
interval). The pairwise kappa value (Table 3) for agreement
among observers ranged from substantial to almost perfect
(0.72 to 0.88). Table 4 shows the proportion of cases
assigned to each category of immunohistochemical score
stratified by the observers. The weak positive category
Am J Clin Pathol 2000;113:852-859
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Hoang et al / HER-2/NEU AMPLIFICATION AND HER-2/NEU OVEREXPRESSION
reflected significant differences in the scoring by the
observers, ranging from 3% to 10%. However, interobserver
variability was reduced when the weak and strong positive
cases were combined. Good agreement was seen in the negative immunohistochemical category, with assigned percentages ranging from 72% to 75%.
The overall concordance rate between gene amplification and protein overexpression was 90% (P < .0001). None
of the 74 cases with negative (0-1+) immunohistochemical
staining, neither of the 2 cases with equivocal staining, 1 of
the 6 cases with weak (2+) immunohistochemical staining,
and 16 of the 18 cases with strong (3+) immunohistochemical staining demonstrated gene amplification by FISH
(Table 5). The prediction of gene amplification based on
immunohistochemical results was 89% for the strong positive
(3+) cases and 17% for the weak positive (2+) cases. Benign
ductal epithelium was present in 54 cases. In contrast with
A
Discussion
Our comparative study shows an association between
gene amplification and protein overexpression with a
concordance rate of 76% for e2-4001 and 91% for the
HercepTest. When the comparison was made in relation to
B
❚Image 1❚ A and B, Two cases showing membranous staining
for HER-2/neu protein scored as 3+ by all 4 observers
(HercepTest, DAKO, Carpinteria, CA). C, Fluorescence in situ
hybridization also showed amplification of HER-2/neu gene
copy levels.
C
856
results seen with the e2-4001 antibody, none of these cases
demonstrated membranous staining. The percentage of positive tumor cells varied from 15% to 100% among the positive cases, and it was more than 75% in all 17 cases that
demonstrated concordant FISH and immunohistochemical
results. ❚Image 1❚ and ❚Image 2❚ illustrate examples of
immunohistochemical studies in which there was complete
as well as partial agreement among raters and their corresponding FISH results.
Am J Clin Pathol 2000;113:852-859
© American Society of Clinical Pathologists
Anatomic Pathology / ORIGINAL ARTICLE
A
C
immunohistochemical scores (negative, 2+, 3+), the correlation between negative protein expression and lack of gene
amplification was highly improved and approached 99%
and 100% for e2-4001 and the HercepTest, respectively.
However, among cases demonstrating protein overexpression, no gene amplification was detected in 21 (57%) of 37
positive cases stained with e2-4001 and 7 (29%) of 24 positive cases stained with HercepTest. Persons et al18 reported
that 7 (7%) of 100 cases demonstrated HER-2/neu protein
overexpression without HER-2/neu gene amplification.
Others reported similar findings, with percentages ranging
from 5%12 to 1%.13 While the differences in these reported
percentages were related partially to the different antibodies being used for immunohistochemical studies and the
different scoring systems, it has been speculated that some
type of transcriptional or posttranscriptional regulation is
responsible for the increased surface receptor expression in
© American Society of Clinical Pathologists
B
❚Image 2❚ A, Membranous staining for HER-2/neu protein
scored as 3+ by 2 observers and 2+ (weak positive) by 2
observers; the consensus score was 2+ (HercepTest, DAKO,
Carpinteria, CA). B, Fluorescence in situ hybridization (FISH)
showing no amplification of gene copy levels. C, Membranous staining for HER-2/neu protein scored as +1 (weak) by 2
observers, negative by 1, and equivocal by 1; the consensus
score was equivocal (HercepTest). Corresponding FISH
showed no amplification of gene copy levels.
the absence of gene amplification in breast carcinoma.19
Pauletti et al20 stated that 3% of breast tumors would overexpress HER-2/neu protein or messenger RNA in the
absence of detectable gene amplification.
Conversely, rare cases have gene amplification without a
detectable level of protein overexpression. In our study, only
1 case with amplified HER-2/neu gene stained by e2-4001
was considered negative for protein overexpression, although
5% of the tumor cells showed 2+ membranous staining.
Similar findings were reported by Persons et al,18 Jacobs et
al,12 and Couturier et al,13 in whose studies 1 (1%) of 100, 2
(2%) of 100, and 1 (1%) of 100 cases with amplified HER2/neu gene demonstrated no protein overexpression, respectively. It is conceivable that preanalytic factors may have
contributed to the discrepant results. Because our study is
composed partly of material submitted on a referral basis, the
effects of tissue fixation and processing cannot be excluded.
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Hoang et al / HER-2/NEU AMPLIFICATION AND HER-2/NEU OVEREXPRESSION
In a recent study by Jacobs et al,12 membranous staining
of the normal ductal epithelium was observed, and the
authors suggested that the final scoring should be based on
subtracting the score of the normal ductal epithelium from
the tumor score. In our study, 3 cases demonstrated membranous staining of the normal epithelium when stained with the
e2-4001 antibody; however, when the immunohistochemical
results were considered negative, the predictive value of the
remaining cases was still low. On the other hand, with the
HercepTest, none of the cases showed positive staining of the
benign epithelium. Therefore, the HercepTest is a more reliable immunohistochemical stain, especially in cases of
metastasis wherein no normal breast epithelium would be
available for evaluation.
Although immunohistochemistry is less technically
challenging and more rapid than FISH, the subjective
measurement of staining intensity and the variability
among various antibodies make FISH a more reliable test
in selected cases. Since more than 50% of invasive breast
carcinomas are HER-2/neu negative, and the negative
predictive value for immunohistochemistry is 99% or more,
it is conceivable that immunohistochemistry should be
considered as a screening test. Because the majority of the
discrepant cases were in the 2+ or weak positive group,
confirmation of these cases by FISH is especially necessary. Clinical trials have shown that disease responds to
Herceptin therapy only in cases with 3+ overexpression; as
a result, most clinical trials involve patients whose tumor
samples display 3+ overexpression.21,22 The lack of clinical
response in 2+ cases on one hand and the lack of correlation with gene amplification on the other (6%-17%)
suggest that weak (2+) overexpression is not specific to
gene amplification, and, therefore, confirmation of these
cases by FISH is necessary before therapy.
We have shown that the HercepTest has better correlation with FISH and less interobserver variability than e24001. However, both e2-4001 and the HercepTest may be
considered good screening tests because in 99% and 100%,
respectively, of the cases with negative (0-1+) staining, FISH
results were concordant. Weak (2+) overexpression of the
HER-2/neu protein especially shows low correlation with
gene amplification for both immunohistochemical stains.
This finding, coupled with the low interobserver reproducibility in separating 2+ from 3+ cases, necessitates
further confirmation of these cases with FISH in the settings
of clinical trials and treatment decisions. Additional prospective studies to assess the predictive value of FISH in conjunction with immunohistochemistry and with patient responses
to Herceptin are warranted.
From the Department of Pathology, the University of Texas M.D.
Anderson Cancer Center, Houston.
858
Am J Clin Pathol 2000;113:852-859
Address reprint requests to Dr Sneige: The University of
Texas M.D. Anderson Cancer Center, Dept of Pathology, Box 53,
1515 Holcombe Blvd, Houston, TX 77030.
Acknowledgments: We thank Thomas Brooks, Raju
Nandagiri, Leslie E. Nesbitt, and Emily Tarco, for technical
assistance and Edward N. Atkinson, MD, for statistical analysis.
References
1. NIH Consensus Conference. Treatment of early-stage breast
cancer. JAMA. 1991;265:391-395.
2. 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.
3. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the
HER-2/neu proto-oncogene in human breast and ovarian
cancer. Science. 1989;244:707-712.
4. Press MF, Berstein L, Thomas L, et al. HER-2/neu gene
amplification characterized by fluorescence in situ
hybridization: poor prognosis in node negative breast
carcinomas. J Clin Oncol. 197;15:2894-2904.
5. Seshadri R, Firgaira FA, Horsfall DJ, et al. Clinical
significance of HER-2/neu oncogene amplification in
primary breast cancer. The South Australian Breast Cancer
Study Group. J Clin Oncol. 1993;11:1936-1942.
6. Popescu NC, King CR, Kraus MH. Localization of the
human erbB-2 gene on normal and rearranged chromosomes
17 to bands q12-21.32. Genomics. 1989;4:362-366.
7. Bargmann CI, Hung M-C, Weinberg RA. The neu oncogene
encodes an epidermal growth factor receptor–related
protein. Nature. 1986;319:226-234.
8. Gullick WJ. The role of the epidermal growth factor
receptor and the c-erbB-2 protein in breast cancer. Int J
Cancer Suppl. 1990;5:55-61.
9. Pegram MD, Lipton A, Hayes DF, et al. Phase II study of
receptor-enhanced chemosensitivity using recombinant
humanized anti-p185HER-2/neu monoclonal antibody plus
cisplatin in patients with HER-2/neu-overexpressing
metastatic breast cancer refractory to chemotherapy
treatment. J Clin Oncol. 1998;16:2659-2671.
10. Graziano C. HER-2 breast assay, linked to Herceptin wins
FDA’s okay. CAP Today. 1998;12:13-16.
11. McNeil C. How should HER2 status be determined? J Natl
Cancer Inst. 1999;91:111.
12. Jacobs TW, Barnes MJ, Yazji H, et al. Comparison of
fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) for the evaluation of HER-2/neu. J
Clin Oncol. 1999;17:1974-1982.
13. Couturier J, Nicolas A, Beuzeboc P, et al. High correlation
between ERBB2 amplification detected by FISH and gene
overexpression detected by immuno-histochemistry in breast
cancers [abstract]. Mod Pathol. 1999;12:18A.
14. Jimenez RE, Wallis T, Tabaczka P, et al. Fluorescent in situ
hybridization (FISH) vs immunohistochemistry (IHC) for
determination of HER-2/neu status in breast cancer
[abstract]. Mod Pathol. 1999;12:23A.
15. Hopman AH, Ramaekers FC, Raap AK, et al. In situ
hybridization as a tool to study numerical chromosome
aberrations in solid bladder tumors. Histochemistry.
1988;89:307-316.
© American Society of Clinical Pathologists
Anatomic Pathology / ORIGINAL ARTICLE
16. Fleiss JG. Statistical Methods for Rates and Proportions. New
York, NY: Wiley; 1981:225-232.
17. Landis JR, Koch GG. The measurement of observer
agreement for categorical data. Biometrics. 1977;33:159-174.
18. Persons DL, Borelli KA, Hsu PH. Quantitation of HER2/neu and c-myc gene amplification in breast carcinoma
using fluorescence in situ hybridization. Mod Pathol.
1997;10:720-727.
19. Earp HS, Dawson TL, Li X, et al. Heterodimerization and
functional interaction between EGF receptor family
members: a new signaling paradigm with implications for
breast cancer research. Breast Cancer Res Treat. 1995;35:
115-132.
20. Pauletti G, Godolphin W, Press MF, et al. Detection and
quantitation of HER-2/neu gene amplification in human
breast cancer archival material using fluorescence in-situ
hybridization. Oncogene. 1996;13:63-72.
© American Society of Clinical Pathologists
21. Ingle BJ, Pritchard K, Shepard L, et al. Initial report of the
SWOG biological correlative study of C-ERB-2 expression
as a predictor of outcome in a trial comparing adjuvant CAF
T with tamoxifen (T) alone [abstract]. Proc ASCO. 1998;17.
Abstract 374.
22. Shak S, Paton V, Bajamonde A. Addition of Herceptin
(humanized anti-HER2 antibody) to first-line chemotherapy
for HER2 overexpressing metastatic breast cancer (HER2
+/MBC) markedly increases anticancer activity: a
randomized, multinational controlled phase III trial
[abstract]. Proc ASCO. 1998;17. Abstract 377.
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