Journal of Cancer Therapy, 2013, 4, 41-48
http://dx.doi.org/10.4236/jct.2013.47A007 Published Online August 2013 (http://www.scirp.org/journal/jct)
41
Detection of HER-2/neu Amplification on Fine Needle
Aspirates of Breast Cancer Using Fluorescence in Situ
Hybridization*
Talaat Iman1#, Sorour Amani2, Abdel-Hadi Mona1
1Departments of Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt; 2Clinical Pathology, Alexandria Univer-
sity, Alexandria, Egypt.
Email: #Iman_talaat@yahoo.com
Received June 3rd, 2013; revised July 5th, 2013; accepted July 12th, 2013
Copyright © 2013 Talaat Iman et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The accuracy of diagnostic assays for HER-2/neu in breast cancer is extremely important as HER-2/neu status is essen-
tial in tailoring adjuvant and/or neoadjuvant treatment in every patient. FNAC is widely practiced in Egypt in preopera-
tive diagnosis of breast cancer for its low cost and high diagnostic accuracy. Since the determination of HER-2/neu
protein expression on cytological preparations was previously found to be unreliable for clinical use, we opted for the
assessment of HER-2/neu status in fine needle aspirates using FISH. The main objective of this study was to evaluate
the reliability of HER-2/neu status assessment by FISH on fine needle aspirates of breast cancers by comparing the re-
sults with IHC and FISH on FFPE tissue sections obtained from corresponding surgically resected specimens. Fine nee-
dle aspirates from 40 breast cancer patients with pathologically confirmed breast cancer were included in the study.
They were submitted for HER-2/neu evaluation by FISH. After surgery, the corresponding FFPE sections were evalu-
ated for HER-2/neu by FISH and by IHC. FNAs from 11 cases proved to be amplified by FISH, while 29 cases were
not amplified. Apart from two cases that showed lack of signals, all specimens evaluated by FISH on the corresponding
FFPE sections showed matched results. The Measurement of Agreement between FISH on FNAs and FISH on FFPE
sections was 86.7%, while that between FISH on FNAs and IHC was 72.5%. The high concordance rate in the present
study between FISH evaluation of HER-2/neu gene amplification on FNAC samples and their corresponding FFPE
samples indicate that FISH may be a reliable technique for HER-2/neu assessment on FNAs. Furthermore, FISH on
FNAs gave us better hybridization signals than their corresponding FFPE tissue sections. Finally, we also conclude that
all score (2+) cases by IHC should be reevaluated by FISH which is crucial for the patient management.
Keywords: Breast Cancer; FISH on FNA; Immunohistochemistry; HER-2/neu
1. Introduction
Breast cancer is one of the major health problems in
Egypt and is by far the most common type of cancer in
Egyptian women [1,2]. The development of breast cancer
is associated with a number of genetic alterations involv-
ing the inactivation of tumour suppressor genes and the
activation of oncogenes. A dominant mechanism, leading
to oncogene activation is the amplification of specific
genomic region [3].
The ERBB2 (HER-2/neu) oncogene is amplified and
overexpressed in about 25% of invasive breast carcino-
mas. In general, ERBB2 amplification confers unfavor-
able prognosis, although its significance is less than that
of the traditional prognostic factors—stage and grade [4].
There are two main methods for establishing HER-2/neu
status in the diagnostic setting. HER-2/neu protein ex-
pression can be detected using immunohistochemistry
(IHC), and HER-2/neu gene amplification can be quanti-
fied using in-situ hybridization (ISH) with either fluores-
cence (FISH) or chromogenic (CISH) probe detection [5].
The relationship between HER-2/neu protein levels and
response to treatment is proportional to the degree of over
expression [6,7]. Protein expression level correlates with
HER-2/neu gene copy number, and IHC has been shown
to be reliable in detecting increased protein expression in
*No conflict of interests. No external funds were received, and all ex-
p
enses were paid by the researchers.
#Corresponding author.
Copyright © 2013 SciRes. JCT
Detection of HER-2/neu Amplification on Fine Needle Aspirates of Breast Cancer Using Fluorescence
in Situ Hybridization
42
cases with a high level of gene amplification [8,9]. How-
ever scoring of HER-2/neu IHC is highly subjective and
inter-observer reproducibility can be problematic, espe-
cially for 2+ cases [10].
FISH is a molecular technique performed on forma-
lin-fixed paraffin-embedded (FFPE) tissues, frozen sec-
tions as well as fine needle aspirate biopsy (FNAB),
which permits the direct visual assessment of gene copy
number in interphase and metaphase nuclei [11]. DNA is
more stable than protein, making FISH less fixation sen-
sitive than IHC [12]. Gene amplification correlates di-
rectly with protein and mRNA levels, and FISH has been
shown to be superior to IHC in predicting clinical out-
come and response to trastuzumab therapy [13]. However,
FISH has some disadvantages, one of them that it is much
more expensive than IHC, although given the high cost of
trastuzumab therapy, cost-effectiveness analysis has sug-
gested that FISH testing all 2+/3+ cases and avoiding
unnecessary treatment of false-positive cases may save
money [14].
Preoperative determination of the HER-2/neu status is
becoming increasingly important to plan for the type of
surgery as well as for neoadjuvant chemotherapy in se-
lected patients. Preoperative HER-2/neu testing can be
done by IHC on FFPE core needle biopsies (CNB). Al-
though the test was found to be successful on CNB [15],
still some obstacles exist: The ASCO/CAP guidelines
recommend that cores entirely involved by retraction ar-
tifacts or crush artifacts should not be used as a sample to
perform/interpret HER-2/neu immunohistochemistry [16]
and the UK recommendations precise that observers
should be aware of the range of common artifacts, in-
cluding edge artifacts, which can be problematic in small
biopsy samples [17].
In Egypt, breast FNAB plays a major role in the diag-
nosis of both palpable and non-palpable breast masses
because cost-efficient health care is a priority in this
country and the less expensive but effective diagnostic
methods are preferred. Furthermore Breast FNAB has
proved to have a very goodspecificity, sensitivity, and
diagnostic accuracy [18]. And therefore in our situation it
would be useful to be able to identify HER-2/neu status
by using FNAB specimens.
Unfortunately, HER-2/neu protein expression on cyto-
logical preparations with any of the fixatives was found
to be insufficiently reliable for clinical use [18]. On the
other hand, several studies have reported successful use
of FISH on FNA cytological material to evaluate HER-
2/neu amplification [19-23]. Yet, few studies compared
the efficacy of FISH on FNA specimens and on corre-
sponding FFPE tissue samples [18].
2. Objectives
The objective of this study was to evaluate the reliability
of HER-2/neu status assessment by FISH on FNAs of
breast cancers by comparing the results with FISH and
IHC done on FFPE tissue sections obtained from corre-
sponding surgically resected specimens. We also aimed
at evaluating concordance rates between IHC HER-2/neu
positive and IHC HER-2/neu negative cases with FISH
amplified and non-amplified cases respectively, in order
to validate the HercepTestTM that is newly implemented
in our laboratory.
3. Material and Methods
Between November, 2011 and May, 2012, fine needle
aspirates were collected from 43 female patients having
an American College of Radiology Breast Imaging Re-
porting and Data System (BIRADS) score of 5 on mam-
mogram and undergoing preoperative confirmation of
malignancy. All patients signed informed consents. Two
FNA samples were subsequently discarded from the
study because they were hypocellular, and one sample
was also excluded because the post-operative biopsy show-
ed an in situ ductal carcinoma.
Fine needle aspirates from the remaining 40 breast
cancer patients were included in the study. They were
submitted for routine diagnostic cytology as well as for
HER-2/neu evaluation by FISH. After surgery, the cor-
responding sections of archival formalin-fixed, paraffin-
embedded tissue were evaluated for HER-2/neu by FISH
and by IHC.
3.1. Sample Preparation
1) FNA sample collection: Preoperative samples were
collected by FNA under ultrasound guidance using a
23-gauge needle. Ultrasound guidance was used in all
cases to ascertain the aspiration of sufficient amount of
cells from within the tumor itself and not from any sur-
rounding breast tissue. The aspirated material was smear-
ed on glass slides, two of which positively charged. The 2
smears on positively charged slides were fixed in metha-
nol/glacial acetic acid (3:1) and stored in 20˚C until
their use for FISH. The remaining slides were fixed in
alcohol for routine H&E staining.
2) FFPE tissue sections: Postoperative tissue biopsies
were fixed in 10% buffered formalin overnight and then
were processed for paraffin embedding. Tissue blocks
were then cut into 4 µm thick sections on positively
charged slides, and incubated overnight at 56˚C.
3.2. HER2/neu Immunohistochemistry
Immunohistochemical analysis was performed manually
on FFPE tissue sections using the FDA-approved Her-
cepTestTM, according to the manufacturer’s instructions.
Briefly, following deparaffinization and rehydration,
Copyright © 2013 SciRes. JCT
Detection of HER-2/neu Amplification on Fine Needle Aspirates of Breast Cancer Using Fluorescence
in Situ Hybridization
43
the tissue sections were subjected to heat-activated epi-
tope retrieval in citrate buffer at 96˚C for 30 minutes.
Blocking of endogenous peroxidase preceded the addi-
tion of the primary anti-HER-2/neu antibody (rabbit an-
tihuman c-erbB-2 A0485, Dako, Copenhagen, Denmark)
at 1/100 dilution. Binding of the primary antibody was
revealed by means of the chromogen substrate, 3,3’-dia-
minobenzidine (DAB). Slides were then counterstained
with hematoxylin. A positive tissue control section was
included in each run of immunohistochemical staining, as
well as a negative control slide in which the primary an-
tibody was omitted.
3.3. Scoring of Immunohistochemical Results
The slides were scored independently by 2 histopatholo-
gists (the first and the third authors) according to the
ASCO & CAP guidelines [15] as follows.
3.3.1. Scor e 3 (Posit i ve)
Uniform intense membrane staining of more than 30% of
invasive tumor cells. A homogeneous (chicken wire) pat-
tern should be present.
3.3.2. Scor e 2 (Equivocal)
Complete membrane staining that is non-uniform or weak
but with obvious circumferential distribution in at least
10% of cells, or intense complete membrane staining in
<30% of tumor cells.
3.3.3. Score 1 (Neg ative)
Weak, incomplete membrane staining in any proportion
of invasive tumor cells, or weak, complete membrane
staining in less than 10% of cells.
3.3.4. Score 0 (Neg ative)
No staining is observed in invasive tumor cells.
Before we started testing the patients included in the
study we retrospectively tested 30 patients with known
HER2/neu FISH status using the FDA-approved Da-
koHercepTestTM for immunohistochemistry. Some tech-
nical issues were slightly modified as well as the inter-
pretation criteria in order to meet the ASCO/CAP guide-
lines recommendations. A concordance rate of 90% was
reached. The 10% of cases that showed lack of agreement
(3 cases) were in the category of 2+ by IHC. All assay
procedures were standardized before starting the new
cases.
3.4. FISH Protocol
1) For cytological specimens: Slides were brought to
room temperature and incubated in 2xSSC at 37˚C for 30
min, gradually dehydrated in alcohol, air dried and proc-
essed by FISH.
2) For FFPE tissue sections: deparaffinization, pre-
treatment, enzyme digestion and fixation of slides were
performed using the Vysis Paraffin Pretreatment Kit
(Vysis) according to the manufacturer’s recommended
protocol.
3.5. Probe Application and Hybridization
Denaturation and hybridization were carried out in a
HYBrite Denaturation/Hybridization System for FISH
(Vysis). All slides were denaturated at 72˚C for 2 min.
HER-2/neu probe mix (10 µl for FFPE sections and 3 µl
for FNAC slides) was added and hybridization took place
at 37˚C for 14 - 18 h. The slides were then washed in
post-hybridization wash buffer at 72˚C for 2 min and
counterstained with DAPI/antifade.
3.6. Evaluation of the FISH Signals
In each case, 100 - 200 well preserved, separately located
interphase cells with clearly visible distinct signals were
scored for HER-2/neu and chromosome 17 signals. FISH
signals were analyzed using BX51/61 Olympus fluores-
cent microscope equipped with a suitable set of filters
including: DAPI single band pass, dual band pass FITC/
TRIC and triple band pass (FITC/TR/DAPI) (Olympus,
UK LTD).
Only single, non-overlapping and intact nuclei were
examined. Nuclei lacking hybridization signals were
excluded from the evaluation. Results were expressed
as a ratio of the numbers of copies of the HER-2/neu
gene to the number of the chromosome 17 centromeric
markers, with a ratio greater than two being considered
amplified. Samples were classified as unamplified when
two copies of HER-2/neu and two copies of chromo-
some 17 were found in the majority of cells and also
when the HER-2/neu to chromosome 17 ratio was lower
than two.
3.6.1. Positive
FISH HER-2/neu/CEP17 ratio 2.2 or greater, or
FISH HER-2/neu gene copy number > 6.0.
3.6.2. Equivocal
FISH HER-2/neu/CEP17 ratio 1.8 - 2.2, or
FISH HER-2/neu gene copy number 4.0 - 6.0.
3.6.3. Negative
FISH HER-2/neu/CEP17 < 1.8, or
FISH HER-2/neu gene copy number < 4.0.
(In conjunction with ASCO & CAP guidelines).
Copyright © 2013 SciRes. JCT
Detection of HER-2/neu Amplification on Fine Needle Aspirates of Breast Cancer Using Fluorescence
in Situ Hybridization
44
4. Results
HER-2/neu was evaluated by FISH on forty primary
breast cancer FNAs and both by FISH and IHC on the
corresponding paraffin sections.
The mean patient age was 42.35 years (range, 22 - 65
years).
All the included cases were diagnosed as infiltrating
ductal carcinoma, NOS. Tumor size, grading and staging
as well as lymph node status are shown in Table 1.
4.1. Patients’ Criteria
The mean patient age was 42.35 years (range, 22 - 65
years). The largest group (21/40 (52.5%)) had Stage II
disease, followed by Stage III (10/40, 25%), then Stage I
(6/40, 15%)
Three (7.5%) of the patients had known distant metas-
tases at the time of the surgery, and 27 cases (67.5%)
were negative for lymph node metastases.
4.2. HER-2/neu FISH on FNAs
Out of the 40 FNAs, 11 (27.5%) were amplified, while 29
(72.5%) were unamplified.
In unamplified cases, two red signals and two green
signals were frequently identified (Figure 1).
Amplified tumors, on the other hand, showed increased
number of the red signals either singly or in clusters (Fig-
ure 2).
As regards the feasibility of FISH on cytological smears,
hybridization was successful in all cases.
Table1. Clinico-pathologic characteristics of the forty breast
cancer cases.
Clinico-pathologic
parameter Number of the cases Percentage
Tumor size
<2 cm
2 - 5 cm
>5 cm
6
16
18
15
40
45
Tumor grading
Well differentiated
Moderately differentiated
Poorly differentiated
4
30
6
10
75
15
Lymph nodes status
Negative
Positive
27
13
67.5
32.5
Distant metastasis
Absent
Present
3
37
7.5
92.7
Tumor staging
I
II
III
IV
6
21
10
3
15
52.5
25
7.5
4.3. HER-2/neu FISH on FFPE Sections
Out of the 40 tumors examined, 9 cases (22.5%) were
amplified (Figure 3), whereas 29 cases (72.5%) showed
no signals amplification (Figure 4). Two cases were non-
evaluable due to lack of signals. Amplification signals
were more often distributed in clusters.
4.4. HER-2/neu Immunohistochemistry
On IHC, out of the same previous 40 tumors 24 (60%)
were negative for the protein expression, while 16 cases
(40%) showed overexpression: ten cases (62.5%) were
scored as (3+) (Figure 5) and six cases (37.5%) as (2+)
(Figure 6).
4.5. FISH on FNAs versus FISH on FFPE
Sections
Apart from the two cases that showed lack of signals on
Figure 1. FISH image of a non-amplified Her 2-neu FNA
breast cancer case showing normal pattern of 2 green and 2
red signals after hybridization with HER 2-neu probe.
(Her2-neu red signals, centromeric green signals).
Figure 2. FISH image of an amplified Her 2-neu FNA breast
cancer case showing increased number of red signals {HER-
2/neu} than green signals {Cen 17}.
Copyright © 2013 SciRes. JCT
Detection of HER-2/neu Amplification on Fine Needle Aspirates of Breast Cancer Using Fluorescence
in Situ Hybridization
45
Figure 3. FISH image of the corresponding FFPE section of
the FNA shown in Figure 2 with amplified HER-2/neu sig-
nals representing cluster pattern. Each tumour cell nucleus
demonstrates HER-2/neu signals/CEP 17 signals ratio of >2.2.
Figure 4. FISH image of the corresponding FFPE section of
the FNA shown in Figure 1 showing non-amplified HER-
2/neu signals. Normal pattern is obtained in most of the
scored cells (2 green {cen 17} and 2 red {HER-2/neu} gene
signals).
Figure 5. Positive immunostaining for HER-2/neu (score 3+).
Uniform intense membrane staining in most invasive tumor
cells is seen (Immunoperoxidase, ×400).
Figure 6. FFPE section showing score 2+ immunostaining
for HER-2/neu, complete but weak membrane staining in
more than 50% of tumor cells (Immunoperoxidase, ×400).
FISH histology, all tumors evaluated by FISH on FNAs
and on corresponding paraffin-embedded sections show-
ed matched results.
The Measurement of Agreement (Concordance rate)
between FISH on FNAs and FISH on FFPE sections was
86.7% as shown in Table 2.
4.6. FISH on FNAs versus IHC
All negative cases (score 0 and 1+) by IHC showed lack
of amplification by FISH on FNAs. All (3+) cases by
IHC were amplified by FISH on FNAs. As for the 6 cases
scored as (2+) by IHC, only one of them showed ampli-
fication by FISH on FNA and the other 5 cases were
unamplified. The Measurement of Agreement (Concor-
dance rate) between FISH on FNAs and IHC was 72.5%
as shown in Table 2.
4.7. FISH on FFPE Sections versus IHC
All negative cases (score 0 and 1+) by IHC showed lack
of amplification by FISH on FFPE sections. Eight of the
(3+) cases by IHC were amplified by FISH on FFPE sec
Table 2. HER-2/neu results comparing FISH on cytological
samples with FISH and IHC on the corresponding histo-
logical sections.
FISH on FNAFFPE sections
FISH IHC
AmplifiedUnamplified Overexpressed
Not
expressed
Amplified 9 2 11 0
Unamplified0 29 5 24
Measurement
of agreement86.7% 72.5%
Copyright © 2013 SciRes. JCT
Detection of HER-2/neu Amplification on Fine Needle Aspirates of Breast Cancer Using Fluorescence
in Situ Hybridization
46
tions. However, 2 of the (3+) cases by IHC were non-
evaluable by FISH on FFPE sections. As for the 6 cases
scored as (2+) by IHC, only one of them showed ampli-
fication by FISH on FFPE sections and the other 5 cases
were unamplified. The Measurement of Agreement (Con-
cordance rate) between FISH on FFPE sections and IHC
was 89%.
5. Discussion
The accuracy of diagnostic assays for HER-2/neu in
breast cancer is extremely important as HER-2/neu status
is not only a prognostic marker but also predictive of
response to chemotherapy, particularly to HER-2/neu-
targeted therapy such as trastuzumab [24]. HER-2/neu
data is also used to predict who will most likely benefit
from doxorubicin, taxanes, tamoxifen and other reagents
[25]. The diagnostic tests most widely used are IHC and
FISH, measuring protein over expression and gene am-
plification, respectively [24].
Furthermore preoperative determination of the HER-
2/neu status is becoming increasingly important to plan
for the type of surgery as well as for neo adjuvant che-
motherapy in selected patients.
FNAC is widely practiced in Egypt for the preopera-
tive diagnosis of breast cancer and the preoperative de-
tection of lymph node metastasis. Clinicians in our coun-
try continue to rely widely on FNAC and consider it an
invaluable diagnostic tool. Core needle biopsy is done
only in certain conditions [17]. Unfortunately, however
the determination of HER2/neu protein expression on
cytological preparations was found to be unreliable for
clinical use [18]. We therefore opted for the assessment
of HER2/neu status in fine needle aspirates using FISH,
which was not tried before in our laboratory. By review-
ing the literature, the feasibility of HER2/neu amplifica-
tion detection by FISH in breast cancer FNAs was evalu-
ated by few studies [18-23].
Our study proves the feasibility of HER2/neu amplifi-
cation detection by FISH on FNA smears as hybridiza-
tion was successful in all cases. From the technical point
of view, FISH signals in cytological smears were more
easily visualized than those on FFPE sections. The pres-
ence of isolated cells in smears made the signals identifi-
cation and scoring easier than in tissue sections. This fact
was previously reported by Klijanienko et al. [23] and by
Bozetti et al. [19] who recommended the use of image
analysis in evaluating histological samples. Furthermore,
in our work FNA samples preparation for FISH didn’t
require pretreatment or enzyme digestion with the ad-
vantage of less expenses and shorter protocol time than
their corresponding FFPE sections. As regards agreement
of FISH on FNA smears of breast cancer cases with FISH
on corresponding FFPE sections, apart from two cases
that showed lack of signals on FISH histology, all tumors
evaluated by FISH on FNAs and on corresponding paraf-
fin-embedded sections showed matched results. A good
concordance rate of 86.7% was obtained in our study. If
the 2 non-evaluable cases in FFPE sections were omitted
from the calculations, then the concordance would be
100%. This is in agreement with Bozetti et al. [19] who
found a concordance rate of 91% between FISH cytology
and FISH histology and with Klijanienko et al. [23] who
obtained a strong correlation (P < 0.001) between FISH
on cytological and histological materials.
Furthermore, in our study, all negative cases (score 0
and 1+) by IHC showed lack of amplification by FISH on
FNAs. All 3+ cases by IHC were amplified by FISH on
FNAs. Similar results were obtained by McManus DT et
al. [21] who detected high amplification by FISH on
FNAs from 3 out of the 15 tumors they examined with
matched diffuse membranous staining by IHC of the
same tumors.
However, a concordance rate of only 72.5% was found
between FISH on FNA smears and IHC in our study.
This is attributed to the 6 cases that were given a score of
(2+) by IHC and which gave variable results by FISH, as
only one of them showed amplification by FISH on FNA
and the other 5 cases were unamplified. In the present
study, a 100% agreement was found between the (3+)
cases by IHC and their corresponding FISH on FFPE
sections except for 2 cases showing complete lack of sig-
nals. Similarly a 100% agreement was found between the
score (0) and (1+) cases by IHC and their corresponding
FISH on FFPE sections. However, the overall concor-
dance rate between FISH on FFPE sections and IHC was
89%, owing to the variable agreement of the score (2+)
cases with their FISH results.
This variable agreement of 2+ cases with gene ampli-
fication was previously encountered in several studies
[18,26-28] emphasizing the importance of reevaluation of
the HER-2/neu status by FISH in all (2+) cases.
Although several studies evaluating FISH and IHC
support the view that FISH has a higher accuracy than
IHC [29-31] and that FISH might be a better predictor of
response to trastuzumab therapy [32,33], we still use IHC
in our laboratory as the first line to triage cases and then
the score (2+) ones are further evaluated by FISH. This is
because of the lower cost of IHC which is covered by the
medical insurance of the patient. Furthermore, IHC is
easier to perform and the FISH equipment is not available
in our routine pathology laboratory. Therefore we tried to
validate the FDA-approved HercepTest that is newly im-
plemented in our laboratory. After modification of some
technical issues and interpretation criteria we compared
the HercepTest results with the FISH results of the same
cases. Only 90% concordance was reached before start-
Copyright © 2013 SciRes. JCT
Detection of HER-2/neu Amplification on Fine Needle Aspirates of Breast Cancer Using Fluorescence
in Situ Hybridization
47
ing this research and 89% with the research cases. The
ASCO/CAP guidelines, however, recommend a mini-
mum of 95% concordance for validation, which we
couldn’t reach due to the variable agreement of score (2+)
cases by IHC. In fact HercepTest has been demonstrated
in several studies to produce significant numbers of false
positives [31,34-36].
In conclusion, the high concordance rate in the present
study between FISH evaluation of HER2/neu gene am-
plification on FNAC samples and their corresponding
FFPE samples indicate that FISH may be a reliable tech-
nique for HER2/neu assessment on FNA samples. This is
of great significance in our institution where preoperative
diagnostic FNAC is widely practiced. Evaluation of
HER2/neu gene amplification on FNAC specimen may
play a role in planning neoadjuvant chemotherapy in
candidate patients, in the management of patients with
advanced or recurrent disease and in deciding the type of
surgery whether conservative or radical in selected pa-
tients. Furthermore, FISH on FNAs gave us better hy-
bridization signals than their corresponding FFPE tissue
sections. Finally, we also conclude that all score (2+)
cases by IHC should be reevaluated by FISH which is
crucial for the patient management. And if our hospital
could afford it, we would recommend the use of FISH as
the primary method for assessment of all FNAs and FFPE
sections of breast cancers to determine the HER-2/neu
status.
REFERENCES
[1] Middle East Cancer Consortium (MECC) Egypt, “The
Gharbiah Population-Based Cancer Registry,” Ministry of
Health and Populations Egypt and Middle East Cancer
Consortium, Cairo, 1999.
[2] National Cancer Institue, “Cairo University, Egypt. Can-
cer Registry 2002-2003,” National Cancer Institue, Cairo.
[3] J. Hannemann, P. Kristel, H. van Tinteren, M. Bontenbal,
Q. G. van Hoesel, W. M. Smit, et al., “Molecular Sub-
types of Breast Cancer and Amplification of Topoisom-
erase II Alpha: Predictive Role in Dose Intensive Adju-
vant Chemotherapy,” British Journal of Cancer, Vol. 95,
No. 10, 2006, pp. 1334-41.
[4] R. B. Dickson, R. G. Pestell and M. E. Lippman, “Cancer
of the Breast,” In: V. Devita, S. Hellman and S. A. Ro-
senberg, Cancer Principles & Practice of Oncology, 7th
Edition, Lippincott, Williams & Wilkins, Philadelphia,
2005, pp. 1595-1654.
[5] R. A. Walker, J. M. Bartlett, M. Dowsett, I. O. Ellis, A. M.
Hanby, B. Jasani, et al., “HER2 Testing in the UK: Fur-
ther Update to Recommendations,” Journal of Clinical
Pathology, Vol. 61, No. 7, 2008, pp. 818-824.
doi:10.1136/jcp.2007.054866
[6] D. Varshney, Y. Y. Zhou, S. A. Geller and R. Alsabeh,
“Determination of HER-2 Status and Chromosome 17
Polysomy in Breast Carcinomas Comparing HercepTest
and PathVysion FISH Assay,” American Journal of Cli-
nical Pathology, Vol. 121, No. 1, 2004, pp. 70-77.
doi:10.1309/FUQH92B039025LHG
[7] H. J. Burstein, L. N. Harris, R. Gelman, S. C. Lester, R. A.
Nunes, C. M. Kaelin, et al., “Preoperative Therapy with
Trastuzumab and Paclitaxel Followed by Sequential Ad-
juvant Doxorubicin/Cyclophosphamide for HER2 Over-
expressing Stage II or III Breast Cancer: A Pilot Study,”
Journal of Clinical Oncology, Vol. 21, No. 1, 2003, pp.
46-53.
[8] M. F. Press, R. S. Finn, D. Cameron, A. Di Leo, C. E.
Geyer, I. E. Villalobos, et al., “HER-2 Gene Amplifica-
tion, HER-2 and Epidermal Growth Factor Receptor
mRNA and Protein Expression, and Lapatinib Efficacy in
Women with Metastatic Breast Cancer,” Clinical Cancer
Research, Vol. 14, No. 23, 2008, pp. 7861-7870.
[9] P. L. Fitzgibbons, D. A. Murphy, D. M. Dorfman, P. C.
Roche and R. R. Tubbs, “Interlaboratory Comparison of
Immunohistochemical Testing for HER2: Results of the
2004 and 2005 College of American Pathologists HER2
Immunohistochemistry Tissue Microarray Survey,” Ar-
chives of Pathology & Laboratory Medicine, Vol. 130, No.
10, 2006, pp. 1440-1445.
[10] A. Rhodes, B. Jasani, E. Anderson, A. R. Dodson and A. J.
Balaton, “Evaluation of HER-2/neu Immunohistoche-
mical Assay Sensitivity and Scoring on Formalin-Fixed
and Paraffin-Processed Cell Lines and Breast Tumors: A
Comparative Study Involving Results from Laboratories
in 21 Countries,” American Journal of Clinical Pathology,
Vol. 11, No. 3, 2002, pp. 408-417.
[11] M. B. Lambros, R. Natrajan and J. S. Reis-Filho, “Chro-
mogenic and Fluorescent in Situ Hybridization in Breast
Cancer,” Human Pathology, Vol. 38, No. 8, 2007, pp.
1105-1022. doi:10.1016/j.humpath.2007.04.011
[12] M. F. Press, G. Sauter, L. Bernstein, I. E. Villalobos, M.
Mirlacher, J. Y. Zhou, et al., “Diagnostic Evaluation of
HER-2 as a Molecular Target: An Assessment of Accu-
racy and Reproducibility of Laboratory Testing in Large,
Prospective, Randomized Clinical Trials,” Clinical Can-
cer Research, Vol. 11, No. 18, 2005, pp. 6598-6607.
[13] H. Yaziji, L. C. Goldstein, T. S. Barry, R. Werling, H.
Hwang, G. K. Ellis, et al., “HER-2 Testing in Breast Can-
cer Using Parallel Tissue-Based Methods,” JAMA Net-
work, Vol. 291, No. 16, 2004, pp. 1972-1977.
[14] L. Arnould, P. Roger, G. Macgrogan, M. P. Chenard, A.
Balaton, S. Beauclair, et al., “Accuracy of HER2 Status
Determination on Breast Core-Needle Biopsies (Immu-
nohistochemistry, FISH, CISH and SISH vs FISH),”
Modern Pathology, Vol. 25, No. 5, 2012, pp. 675-682.
[15] A. C. Wolff, M. E. Hammond, J. N. Schwartz, K. L. Ha-
gerty, D. C. Allred, R. J. Cote, et al., “American Society
of Clinical Oncology/College of American Pathologists
Guideline Recommendations for Human Epidermal Growth
Factor Receptor 2 Testing in Breast Cancer,” Journal of
Clinical Oncology, Vol. 25, No. 1, 2007, pp. 118-145.
doi:10.1200/JCO.2006.09.2775
[16] I. O. Ellis, J. Bartlett, M. Dowsett, S. Humphreys, B.
Copyright © 2013 SciRes. JCT
Detection of HER-2/neu Amplification on Fine Needle Aspirates of Breast Cancer Using Fluorescence
in Situ Hybridization
Copyright © 2013 SciRes. JCT
48
Jasani, K. Miller, et al., “Best Practice No 176: Updated
Recommendations for HER2 Testing in the UK,” Journal
of Clinical Pathology, Vol. 57, No. 3, 2004, pp. 233-237.
doi:10.1136/jcp.2003.007724
[17] M. Abdel-Hadi, G. F. Abdel-Hamid, N. Abdel-Razek and
R. K. Fawzy, “Should Fine-Needle Aspiration Cytology
Be the First Choice Diagnostic Modality for Assessment
of All Nonpalpablebreast Lesions? The Experience of a
Breast Cancer Screening Center in Alexandria, Egypt,”
Diagnostic Cytopathology, Vol. 38, No. 12, 2010, pp. 880-
889. doi:10.1002/dc.21305
[18] B. G. Beatty, R. Bryant, W. Wang, T. Ashikaga, P. C.
Gibson, G. Leiman, et al., “HER-2/neu Detection in Fine-
Needle Aspirates of Breast Cancer: Fluorescence in Situ
Hybridization and Immunocytochemical Analysis,” Ame-
rican Journal of Clinical Pathology, Vol. 122, No. 2, 2004,
pp. 246-255.
[19] C. Bozzetti, R. Nizzoli, A. Guazzi, M. Flora, C. Bassano,
P. Crafa, et al., “HER-2/neu Amplification Detected by
Fluorescence in Situ Hybridization in Fine Needle Aspi-
rates from Primary Breast Cancer,” Annals of Oncology,
Vol. 13, No. 9, 2002, pp. 1398-1403.
doi:10.1093/annonc/mdf217
[20] A. Mezzelani, L. Alasio, C. Bartoli, M. G. Bonora, M. A.
Pierotti, F. Rilke, et al., “c-erbB2/neu Gene and Chro-
mosome 17 Analysis in Breast Cancer by FISH on Ar-
chival Cytological Fine-Needle Aspirates,” British Jour-
nal of Cancer, Vol. 80, No. 3-4, 1999, pp. 519-525.
[21] D. T. McManus, A. H. Patterson, P. Maxwell, M. W.
Humphreys and N. H. Anderson, “Fluorescence in Situ
Hybridisation Detection of erbB2 Amplification in Breast
Cancer Fine Needle Aspirates,” Molecular Pathology, Vol.
52, No. 2, 1999, pp. 75-77.
[22] G. Sauter, G. Feichter, J. Torhorst, H. Moch, H. Novotna,
U. Wagner, et al., “Fluorescence in Situ Hybridization for
Detecting erbB-2 Amplification in Breast Tumor Fine
Needle Aspiration Biopsies,” Acta Cytologica, Vol. 40,
No. 2, 1996, pp. 164-173.
[23] J. Klijanienko, J. Couturier, M. Galut, A. K. El-Naggar, Z.
Maciorowski, E. Padoy, et al., “Detection and Quantita-
tion by Fluorescence in Situ Hybridization (FISH) and
Image Analysis of HER-2/neu Gene Amplification in
Breast Cancer Fine-Needle Samples,” Cancer, Vol. 87,
No. 5, 1999, pp. 312-318.
[24] A. M. Gown, “Current Issues in ER and HER2 Testing by
IHC in Breast Cancer,” Modern Pathology, Vol. 21, No.
S8-S15, 2008, p. 34.
[25] A. Thor, “Applications of Molecular Analysis to Small
Biopsy and Cytology Specimens for Diagnosis and Tar-
geted Therapy. HER2/neu (erbB2) Analysis on FNA
Smears and Core Biopsies of Primary or Metastatic Breast
Cancer,” PSC/USCAP, 2008.
http://pathologyportal.org/97th/pdf/companion16h03.pdf
[26] M. F. Press, D. J. Slamon, K. J. Flom, J. Park, J. Y. Zhou
and L. Bernstein, “Evaluation of HER-2/neu Gene Am-
plification and Overexpression: Comparison of Frequently
Used Assay Methods in a Molecularly Characterized Co-
hort of Breast Cancer Specimens,” Journal of Clinical
Oncology, Vol. 20, No. 14, 2002, pp. 3095-3105.
[27] K. I. Goud, S. Dayakar, K. Vijayalaxmi, S. J. Babu and P.
V. Reddy, “Evaluation of HER-2/neu Status in Breast
Cancer Specimens Using Immunohistochemistry (IHC) &
Fluorescence In-Situ Hybridization (FISH) Assay,” In-
dian Journal of Medical Research, Vol. 135, No. 3, 2012,
pp. 312-317.
[28] R. D. Mass, M. F. Press and S. Anderson, “Improved
Survival Benefit from Herceptin (Trastuzumab) in Pa-
tients Selected by Fluorescence in Situ Hybridization
(FISH) (Abstract),” Proc Am Soc Clin Oncol, Vol. 20, No.
22a, 2001.
[29] S. Kakar, N. Puangsuvan, J. M. Stevens, R. Serenas, G.
Mangan, S. Sahai, et al., “HER-2/neu Assessment in Breast
Cancer by Immunohistochemistry and Fluorescence in
Situ Hybridization: Comparison of Results and Correla-
tion with Survival,” Journal of Molecular Diagnostics,
Vol. 5, No. 3, 2000, pp. 199-207.
[30] M. F. Press, L. Bernstein, P. A. Thomas, L. F. Meisner, J.
Y. Zhou, Y. Ma, et al., “HER-2/neu Gene Amplification
Characterized by Fluorescence in Situ Hybridization: Poor
Prognosis in Node-Negative Breast Carcinomas,” Journal
of Clinical Oncology, Vol. 15, No. 8, 1997, pp. 2894-
2904.
[31] G. Pauletti, S. Dandekar, H. Rong, L. Ramos, H. Peng, R.
Seshadri, et al., “Assessment of Methods for Tissue-
Based Detection of the HER-2/neu Alteration in Human
Breast Cancer: A Direct Comparison of Fluorescence in
Situ Hybridization and Immunohistochemistry,” Journal
of Clinical Oncology, Vol. 18, No. 21, 2000, pp. 3651-
3664.
[32] C. L. Vogel, M. A. Cobleigh, D. Tripathy, J. C. Gutheil, L.
N. Harris, L. Fehrenbacher, et al., “Efficacy and Safety of
Trastuzumab as a Single Agent in First-Line Treatment of
HER2-Overexpressing Metastatic Breast Cancer,” Jour-
nal of Clinical Oncology, Vol. 20, No. 3, 2002, pp.
719-726.
[33] M. A. Cobleigh, C. L. Vogel, D. Tripathy, N. J. Robert, S.
Scholl, L. Fehrenbacher, et al., “Multinational Study of
the Efficacy and Safety of Humanized Anti-HER2 Mono-
clonal Antibody in Women Who Have HER2-Overex-
pressing Metastatic Breast Cancer That Has Progressed
after Chemotherapy for Metastatic Disease,” Journal of
Clinical Oncology, Vol. 17, No. 9, 1999, pp. 2639-2648.
[34] T. W. Jacobs, A. M. Gown, H. Yaziji, M. J. Barnes and S.
J. Schnitt, “Specificity of HercepTest in determining HER-
2/neu Status of Breast Cancers Using the United States
Food and Drug Administration-Approved Scoring Sys-
tem,” Journal of Clinical Oncology, Vol. 17, No. 7, 1999,
pp. 1983-1987.
[35] P. C. Roche and J. N. Ingle, “Increased HER2 with US
Food and Drug Administration-Approved Antibody,”
Journal of Clinical Oncology, Vol. 17, No. 1, 1999, p.
434.
[36] A. Lebeau, D. Deimling, C. Kaltz, A. Sendelhofert, A. Iff,
B. Luthardt, et al., “Her-2/neu Analysis in Archival Tis-
sue Samples of Human Breast Cancer: Comparison of
Immunohistochemistry and Fluorescence in Situ Hy-
bridization,” Journal of Clinical Oncology, Vol. 19, No. 2,
2001, pp. 354-363.