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Luminiţa Leluţiu1, Rareş Buiga1, Liliana Resiga1, Alexandru Eniu1, Nicolae Ghilezan2, Corneliu D. Olinici2
1
Ion Chiricuţă Cancer Institute, Cluj-Napoca, Romania 2Iuliu Hatieganu University of Medicine and Pharmacy ClujNapoca, Romania
Background: The sample was chosen to contain all situations possible as in the guidance for authors. You can copy this
sample and modify inside by Copy from your original document and Edit/Paste special/Unformatted text. Only documents provided in Word 2003 will be accepted. Tables were chosen on one column or on two column. Patients and
methods: The authors compared the specificity, simplicity of a procedure and method standardization, the simplicity of
evaluation for the results for each method (immunohistochemistry and chromogenic in situ hybridization). 1255 cases
of invasive breast carcinoma from surgically excised tumors and core needle biopsies were included in the study. The
first step was the determination the HER2 status by immunohistochemistry. The cases with moderate (2+) and strong
(3+) overexpression of HER2 protein were chosen for determining HER2 amplification by CISH. Results: Falsepositive results are a significant problem when IHC is exclusively used to test for HER2 overexpression. The falsepositives results are confined to the group 2+ positive and do not respond to targeted therapy. Concordance between
IHC and CISH is high when immunostaining is interpreted as either negative or strongly positive. Many studies have
suggested that CISH may better predict response to anti-HER2 therapy rather than IHC. Although IHC is less technically demanding it is more expensive than CISH. Conclusions: As a first step, IHC analysis of HER2 status in breast cancer is a useful predictor of response to therapy with Herceptin when it is strongly positive. In some cases IHC positive
overexpressors are false-positives in CISH tests. Thus, screening of breast cancer with IHC and then confirmation 2+
positives IHC results by CISH is an effective evolving strategy for testing HER2 as a predictor of response to targeted
therapy.
Key words: Laryngeal Cancer, Lymph Node Involvement, Postoperative Radiotherapy.
Introduction1
Gene amplification is frequently detected in
human tumor cells and is thought to make an important
contribution to tumorigenesis. Detailed analysis of altered regions of DNA has revealed complex DNA rearrangements often involving multiple genes and spanning several megabases in solid tumors. Many tumors
show such a high degree of general DNA and chromosome rearrangement that some researchers argue the
critical event in tumorigenesis is genomic instability
with gene amplification being a consequence.
The human epidermal growth factor receptor-2
gene ERBB2 (commonly referred to as HER2/neu) is
located on the long arm of chromosome 17q and encodes a transmembrane tyrosine kinase receptor with
extensive homology to the epidermal growth factor receptor (EGFR) or HER family.This family of receptors
Journal of Radiotherapy & Medical Oncology
March 2009 Vol. XV No. 1:29-35
Address for correspondence: Luminiţa Leluţiu,
Ion Chiricuţă Cancer Institute,
34-36 Republicii Street, 400015
Cluj-Napoca, Romania
email: luminita_lelutiu@yahoo.com
is involved in cell-to-cell and cell-to-stroma communication primarly through a process known as signal
transduction.
HER2/neu gene amplification and/or protein
overexpression has been identified in 18-20% of invasive breast cancer (early studies suggested that as many
as 30% of breast cancers have HER2 overexpression).The presence of HER2/neu gene amplification is
prognostically and therapeutically significant for patients
with breast cancer. Overexpression of HER2 protein is
associated with rapid tumour growth, increased risk of
recurrence after surgery, poor response to conventional
chemotherapy and shortened survival.
Testing to determine HER2 status has come into
focus since the approval of transtuzumab (Herceptin) for
the treatment of HER2-positive breast cancer.
Transtuzumab (Herceptin) is a monoclonal antibody directed specifically against HER2 protein and was approved in 1998 by the US Food and Drug Administration for the treatment of metastatic disease. The benefit
has been evidenced when transtuzumab is used with
chemotherapy as a first treatment for metastatic breast
cancer or as a single agent for patients with metastatic
disease that progresses after initial chemotherapy.
The most commonly used methods for HER2
testing are immunohistochemistry (IHC), which
measures HER2 protein in the cell membrane, and fluorescence in situ hybridization (FISH) or chromogenic in
situ hybridization (CISH), which evaluates HER2/neu
gene amplification. Both methods are able to evaluate
HER2/neu in formalin–fixed paraffin-embedded archival tissues.
Most HER2/neu studies have been performed
by IHC, which detect overexpression of the HER2 protein product on the cell membrane of tumor cells. The
technique is widely accessible and easy to perform at a
reasonable cost. This semi-quantitative method is beset
by technical artifacts, sensitivity differences between
different antibodies, and subjective interpretation, resulting in interobserver variability between pathologists. For
IHC stain intensity 2+ additional testing, with FISH or
CISH is recommended.
FISH is a quantitative procedure for detecting
HER2/neu gene amplification on the nuclei of tumor
cells. The FISH method is a more complex and more
expensive technique; a disadvantage of FISH is the requirement of expensive equipment for the analysis(a
fluorescent microscope with suitable filters, a sensitive
CCD camera).Moreover, fluorescent signals tend to fade
in a few weeks or months and it does not allow for a
simultaneous histological examination of tissue structure
beyond basic tissue identification.
CISH is a new technique used in HER2/neu
gene detection. The concordance between CISH and
FISH can range from 85% to as high as 100%. The advantages of CISH are: it requires an ordinary microscope; the method is more economical; the signal intensity is permanent.
Patients and methods
Our study was focused on 1255 breast carcinoma tissue samples with final histopathologic diagnosis
of invasive ductal, lobular carcinoma and other special
types. From a total number of 1255 cases, 960 cases
were diagnosed and treated between January 2006 and
September 2008 at the Ion Chiricuţă Cancer Institute
Cluj-Napoca and 295 samples were received from other
hospitals only for HER2 testing by IHC or CISH.
All 960 tumors were removed by mastectomy
or core needle biopsies (breast-conserving therapy), and
axillary dissection was performed.
The patients ages were grouped as either
premenopausal (less than 50 years) or postmenopausal
(50 years and more). The age of the patients with breast
cancer ranged from 26 to 81 years with a median age of
52 years.
Histologic typing was performed according to
the World Health Organization classification and grading according to the Elston and Ellis modified criteria of
Bloom and Richardson (Table I).
The pathologists reviewed the HER2 results of
all IHC cases in the period covered, reassessing them in
accordance with the US Food and Drug Administrationapproved HER2 Herceptin scoring guidelines.
Immunohistochemical Analysis
For each case, all H&E-stained slides were reviewed and 1 or 2 representative tumor tissue blocks
were selected for study. Immunohistochemical analysis
were performed on 4 μm sections according to standard
procedures. We cut 4 μm sections from archival tissue
that had been fixed in buffered formalin and embedded
in paraffin, and mounted on silanized slides (SuperFrost
Plus).
The IHC-stained slides were interpreted and
scored on the scale of 0 to 3+ according to the FDAapproved guidelines for HercepTest (Table II). Areas
with intraductal carcinoma were excluded from the
evaluation.
Some cases with 2+ and 3+ IHC results were
further evaluated by CISH, for HER2/neu gene amplification.
Our laboratory participates regularly in an international UK-based quality assurance system
(NEQUAS) for immunohistochemistry.
Luminiţa Leluţiu et al: HER2 Testing in Breast Cancer Using Immunohistochemical Analysis…
31
Table I: Tumor and histological characteristics
Tumor histological grade
Ductal
Age
< 50
≥ 50
Total ductal
Lobular
< 50
≥ 50
Total lobular
Ductal+Lobular
< 50
≥ 50
Total D+L
Mucinous
< 50
≥ 50
Total mucinous
Apocrine
< 50
≥ 50
Total apocrine
Micro
< 50
papillary
≥ 50
Total micro
Grand total
G1
No
53
73
126
5
14
19
2
3
5
1
4
5
0
0
0
0
0
0
155
G2
%
5.52
7.60
13.13
0.52
1.46
1.98
0.21
0.31
0.52
0.10
0.42
0.52
0
0
0
0
0
0
16.15
No
144
247
391
6
11
17
9
14
23
0
0
0
0
0
0
1
0
1
432
G3
%
15.00
25.73
40.73
0.63
1.15
1.77
0.94
1.46
2.40
0
0
0
0
0
0
0.10
0
0.10
45.00
No
131
212
343
3
1
4
4
18
22
0
1
1
2
1
3
0
0
0
373
%
13.65
22.08
35.73
0.31
0.10
0.42
0.42
1.88
2.29
0
0.10
0.10
0.21
0.10
0.31
0
0
0
38.85
Grand total
No
%
328
34.17
532
55.42
860
89.58
14
1.46
26
2.71
40
4.17
15
1.56
35
3.65
50
5.21
1
0.10
5
0.52
6
0.63
2
0.21
1
0.10
3
0.31
1
0.10
0
0
1
0.10
960
Table II: Recommended immunohistochemical scoring method
Score
0
HER2 protein assesstment
Negative
1+
Negative
2+
Equivocal
3+
Positive
Staining pattern
No staining is seen or membrane staining is less that 10% invasive
tumour cells
Faint/barely perceptive membrane staining detected in more than
10% invasive tumour cells
Weak to moderate complete membrane staining in more than 10%
invasive tumour cells or < 30% with strong complete membrane
staining
Strong, uniform complete membrane staining in more than 30%
invasive tumour cells
Chromogenic In Situ Hybridization (CISH)
For chromogenic in situ hybridization standardization (CISH) analysis, we selected 56 cases with
moderate (2+) and strong (3+) overexpression of
HER2 protein detected by IHC in our laboratory and in
another hospital center, which were clinically eligible
for treatment with transtuzumab.
We cut 4 μm sections from the archival tissue
that had been fixed in buffered formalin and embedded
in paraffin. Then, the sections were deparaffinized and
heated in CISH pretreatment buffer (SPOT-Light tissue
pretreatment kit, Zymed Laboratories). Immunodetection was performed by using a commercial SPOT-
Light HER2 CISH Detection Kit (Zymed) according to
the manufacturer’s instructions.
HER2 gene signals were scored in histologic
sections in at least 200 neoplastic cells with a microscope Nikon E-1000 using a 40x objective.
The CISH assays were run by one technologist
following the protocol of Zymed. CISH results were
read by the same pathologist who was blinded to the
previous IHC results.
CISH was assessed on invasive ductal carcinoma areas only; foci of intraductal carcinoma were
excluded from the analysis.
Tumors were classified depending on the
number of gene copies in the nuclei as follows Table
III (the recommended CISH scoring by Zymed):
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Journal of Radiotherapy & Medical Oncology, March 2009 Vol. XIV No. 1:29-35
Tabel III: Evaluation of HER2 gene status using Chronogenic in Situ Hybridization (CISH)
Amplification
HER2/neu gene status
High-level amplification
> 10 dots, or large cluster, or mixture of multiple dots and
large clusters, of HER2 gene per nucleus in > 50% tumor
cells
6-10 dots, or small clusters, or mixture of multiple dots and
small clusters of HER2 gene per nucleus in > 50% tumor
cells
Polysomy: 3-5 dots of HER2 per nucleus of 50% tumor cells
Diploid: 1-2 dots of HER2 per nucleus of 50% tumor cells
Low-level amplification
No amplification
Imunohistochemical (IHC) Results
A total of 1255 breast cancer specimens were
tested for HER2 status: 960 (76.49%) from the Cancer
Institute and 295 (23.51%) from other hospitals. From
these 960 cases, 860 (89.58%) were invasive ductal
carcinoma (126 cases with SBR grade 1, 391 cases
with SBR grade 2, and 343 cases with SBR grade 3),
40 (4.17%) were invasive lobular carcinoma, 50
(5.21%) were invasive ductal + lobular carcinoma and
10 cases (1.04%) other special types (mucinous carcinoma, invasive micropapillary carcinoma, apocrine
carcinoma).
The 591 (61.56 %) of 960 specimens evaluated
with the HercepTest were scored 0,154 (16.04%) were
scored 1+, 75 (7.81%) were scored 2+ and 140
(14.58%) were scored 3+ (Table IV).
Table IV: Imunohistochemcal Analysis of Breast Tumor Specimens for HER2 Testing
IHC score
0
1+
2+
3+
No. (%) (N=960)
591(61.56)
154 (16.04)
75 (7.81)
140 (14.58)
All 40 cases with final histopathological diagnosis of invasive lobular carcinoma were scored negative (0,1+) by IHC.
All cases (295) from other hospitals were invasive ductal carcinoma: 61 cases (20.67%) were IHC
positive (2+, 3+) and 234 (79.32%) were IHC negative
(0, 1+).
IHC positive specimens (invasive ductal carcinoma with 2+ and 3+ IHC results) were futher evaluated by CISH (56 cases in our laboratory).
Table V shows the IHC assay in relation to the
tumor histologic grades.Of the 960 cases, 215
(22.39%) were IHC positive and 745 (77.60%) were
IHC negative. From the Cancer Institute most of the
tumors IHC positive tumors were invasive ductal carcinoma with histologic grade 3 (50.23%), followed by
grade 2 (42.79%), then grade 1 (6.97%). Most of the
patients with histologic grade 3 and 2 were in postmenopausal status (50 years or older). No significant
association was noted between histologic grade and
IHC scores (P = 0.928). Similarly, no association was
observed between age and histologic grade (P=0.929).
Many patients more than 50 years old and older (postmenopausal status) seemed to be HER2 positive and amplified, whereas more patients less than 50
years old (premenopausal status) seemed to be HER2
negative and non-amplified (Table VI).However, these
associations were not statistically significant (P=0.962)
Chronogenic in Situ Hybridization (CISH) results
All 56 tests were successfully performed with
the Zymed protocol for CISH.At least two detectable
amplicons per tumor nucleus were observed, and these
served as the internal control.In addition, a highly amplified positive control was run simultaneously with
each test.
CISH result were evaluated with the light microscope at low-power and high-power magnification.
Amplified cases showed at least six brown signals per
nucleus; distinct clustering of amplicons was also obtained. Those cases reported as having no amplification
or low amplification were not verified further for
chromosome 17 aneuploidy.
Table VII shows the CISH assay outcomes of
the 56 cases (cases from our laboratory with 2+ and 3+
IHC results and cases that were tested by IHC in other
center) in relation to the IHC category.Of the 56 cases,
36 were IHC 2+ positive and 20 were IHC 3+ positive.With the CISH assay, 23 (66.66%) of the 36 cases
with IHC 2+ showed HER2/neu gene amplification,
and 12 (33.33%) no amplification. All 20 cases with
IHC 3+ showed high amplification with CISH assay.
Table V: Breast tumor samples and their corresponding immunohistochemistry (IHC) and tumor histologic grade
IHC result
Age
G1
G2
G3
Grand total
HER 0
< 50
≥ 50
47 (21.86%)
77 (20.48%)
124 (20.98%)
5 (8.33%)
11 (11.70%)
16 (10.39%)
7 (20.00%)
2 (5.00%)
9 (12.00%)
2 (3.92%)
4 (4.49%)
6 (4.29%)
155 (16.15%)
86 (40.00%)
172 (45.74%)
258 (43.65%)
35 (58.33%)
47 (50.00%)
82 (53.25%)
12 (34.29%)
15 (37.50%)
27 (36.00%)
27 (52.94%)
38 (42.70%)
65 (46.43%)
432 (45.00%)
82 (38.14%)
127 (33.78%)
209 (35.36%)
20 (33.33%)
36 (38.30%)
56 (36.36%)
16 (45.71%)
23 (57.50%)
39 (52.00%)
22 (43.14%)
47 (52.81%)
69 (49.29%)
373 (38.85%)
215
376
591
60
94
154
35
40
75
51
89
140
960
Total HER 0
HER 1+
< 50
≥ 50
Total HER 1+
HER 2+
< 50
≥ 50
Total HER 2+
HER 3+
< 50
≥ 50
Total HER 3+
Grand total
Tabel VI: Comparison of age (menopausal status) and immunohistochemistry results
Parameter
Immunohistochemistry
Negative (0, 1+)
Positive (2+)
Positive (3+)
Total
Age <50
Age ≥ 50
Total
275 (36.91%)
35 (46.66%)
51 (36.42%)
361
470 (63.08%)
40 (53.33%)
89 (63.57%)
599
745
75
140
960
Tabel VII: Chromogenic in situ hybridization (CISH) results
IHC
Total cases
No amplification
With amplification
• high-level
• low-level
Score 2+
36
12 (33.33%)
24 (66.66%)
9
15
Discussion
In the present study, 276 (21.99%) of 1255
cases were IHC positive and 979 (78%) were IHC
negative.
From the Cancer Institute most of the IHC
positive tumors were invasive ductal carcinoma with
histologic grade 3 (50.23%), followed by grade 2
(42.79%), then grade 1 (6.97%). Most of the patients
with histologic grade 3 and 2 were in postmenopausal status (50 years or older).
Histological grading by itself is not accepted
as a prognosis factor. However, several studies have
proved that 3+ histological graded is associated with
IHC markers, related to poor prognosis.
All 40 cases with final histopathological diagnosis of invasive lobular carcinoma were scored
negative (0,1+) by IHC, and these results are in concordance with the previous reports.
From total IHC positive results, 56 tests selected were successfully performed with the Zymed
Score 3+
20
0
20 (100%)
20
0
protocol for CISH.The concordance between 3+ IHC
and CISH-amplified cases was 100% (20 out 20),
denoting all gene amplified cases to be overexpressing the HER2 protein.In contrast, the agreement on
2+ IHC and CISH-amplified cases was only 66.66%,
which is lower than previously reported in other
study. The agreement on IHC positive and CISHamplified cases was 78.57%. The 33.33% (12 of 36)
cases IHC-positive/CISH-non-amplified tumors in
this study, all of which had 2+ IHC scores, is higher
than the 17% false positive reported by the literature.
In this case, false-positive results are a significant
problem when IHC is exclusively used to test for
HER2 overexpresion. A significant percentage (515%) of breast carcinomas show moderate HER2
membrane staining without evidence of amplification, and they most represent false positive staining
(highly sensitive nonspecific staining). It may be
caused by alternative transcriptional or posttranscriptional mechanisms controlling HER2 expression.
Fig. 1: HER2 protein testing in breast cancer (IHC) : (a) No staining is seen (score=0);
(b) Faint perceptive membrane staining detected in more than 10% invasive tumour cells (score=1+);
(c) Weak to moderate complete membrane staining in more than 10% invasive tumour cells (score=2+);
(d) Strong, uniform complete membrane staining in more than 30% invasive tumour cells(score=3+)
Fig. 2: Chromogenic in situ hybridization (CISH) analysis shows: (a) One or two dots can be seen in each nucleus,
indicating the absence of HER2/neu gene amplification.(b) Small clusters and multiple dots can be seen, indicating a low
level amplification of HER2/neu gene.(c) and (d).Large clusters of hybridised HER2 probe, indicating the presence of a high
level of HER2/neu gene amplification.
Some cases, received from other hospitals
that were scored with moderate overexpression (2+)
had in fact the score 1+ (negative). This aspect can
explain discrepancies between the IHC and CISH results in our study.
A test for chromosome 17 aneuploidy was
not performed on non-amplified or low-amplified
CISH assay, because recent studies have shown that
it makes the analysis more costly and time consuming without adding relevant data.
Conclusions
In this study HER2 protein by IHC and
HER2/neu gene amplifications by CISH was successfully evaluated as a molecular biology procedure
that is easily integrated into routine testing in our laboratory. This method is a practical alternative to
FISH that can be used in conjunction with IHC,
which remains the first screening procedure of
choice.
Immunohistochemistry is easy to perform, a
relatively inexpensive method and able to detect the
majority of breast cancer patients whose tumor has a
IHC-negative score (0, 1+) or a 3+ IHC score, all of
which have complete concordance with CISH.
Because discrepancies between IHC and
CISH were observed in the 2+ IHC category, all
cases with 2+ IHC results will be tested by CISH before transtuzumab therapy. This association IHCCISH not only identifies the IHC false-positive subset that will not benefit from transtuzumab, but also
keeps testing costs for HER2 status at a reasonable
minimum.
It is very important that the HER 2 testing by
IHC and CISH is performed in the same laboratory,
to exclude the IHC false-positive/CISH negative results.
CISH is emerging as a practical, costeffective, and valid alternative to FISH in testing for
gene alteration, especially in centers primarily working with IHC.
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The authors ndicated no potential conflicts of interest
Author Contributions:
Conception and design: Luminiţa Leluţiu, C.D. Olinici
Provision of study material or patients: Luminiţa Leluţiu, Rareş Buiga, Alex. Eniu
Collection and assembly of data: Luminiţa Leluţiu
Data analysis and interpretation: Luminiţa Leluţiu, N. Ghilezan
Manuscript writing: Luminiţa Leluţiu
Final approval of manuscript: C.D. Onilici