Can GATA3 Immunocytochemistry be Utilized as a Reliable Diagnostic Marker for Metastatic Breast Carcinoma in Cytological Materials?
A Comparative Study with Mammaglobin and GCDFP-15 Expression
Nesreen H. HAFEZ , HebatAllah M. SHAABAN
Department of Pathology, Cairo University, National Cancer Institute, Cairo, EGYPT
Keywords: GATA 3, Immunostaining, Metastatic breast carcinoma, Cytology
Cytomorphologic differentiation of metastatic breast carcinoma from non breast metastases in cytological materials can be difficult.
Current breast immunocytochemical markers have low sensitivities. Transcription factor GATA 3 is a promising marker for detecting breast
differentiation in cytological materials. The aim of the study was to assess the diagnostic value of GATA 3 as a breast differentiation marker in
metastatic cytological materials and to compare it with expression of mammaglobin and gross cystic disease fluid protein-15 (GCDFP-15).
Material and Method: We retrospectively retrieved 133 cases of metastatic breast carcinoma from the archive the of Cytology Unit between
December 2013 and June 2015. They included 77 fine needle aspiration and 56 serous effusion samples. Forty-five cytological materials from non
mammary metastatic tumors were used as a control. Immunostaining was performed on cell blocks for the presence of GATA 3, mammaglobin
Results: GATA 3 nuclear staining was detected in 82.7% of metastatic breast carcinomas, and 11.1% of metastatic non mammary adenocarcinomas
(p < 0.001). GATA 3 sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 82.7%, 88.9%, 95.7%, 63.5% and
84.3%, respectively. Mammaglobin and GCDFP-15 staining of metastatic breast carcinoma cases was positive in 70.7% and 47.1%, respectively.
GATA 3 staining was significantly higher compared with mammaglobin and GCDFP-15 (p< 0.001).
Conclusion: GATA 3 is more sensitive marker than mammaglobin and GCDFP-15 for diagnosing metastatic breast carcinoma in cytological cell
block materials. Adding mammaglobin to GATA 3 resulted in improvement in its sensitivity. GATA 3 was occasionally positive in some metastatic
non mammary carcinoma that may cause misdiagnosis.
About one third of patients with breast carcinoma have
evidence of metastatic spread during the course of
disease. Although the exact incidence of metastatic breast
carcinoma has not been estimated, it has been reported
that 162 thousand females in the United States lived with
metastatic disease in 2013. This was assessed by oncologists
who encountered cases of metastatic tumor in patients
with breast cancer 1
. In the majority of metastatic cases,
a history of primary tumor is well known. However,
some cases initially present with metastases of unknown
primary. It is very important to identify the primary site
of origin to apply the optimal therapy 2
. As breast cancer
is one of the commonest malignancies affecting females
worldwide, primary breast carcinoma usually enter the differential diagnosis of metastatic carcinoma in females,
even in the absence of breast complaints 3
between metastatic breast and non breast carcinomas
in Papanicolaou-stained cytological slides can be a
challenging mission due to lack of histological architecture
and low cellularity. Immunocytochemical (ICC) markers
can more accurately confirm breast differentiation.
Currently used markers, mammaglobin and gross cystic
disease fluid protein-15 (GCDFP-15) are specific but have
variable sensitivity results (4). GATA3 is one of six zincfinger
transcription factors. It is vital for proliferation and
differentiation of several tissues and is expressed in many
. Recently, GATA3 was reported to be a very
sensitive marker for breast carcinomas 6
GATA3 level has been associated with a worse outcome 7
To our knowledge, the number of studies that evaluate
the diagnostic utility of GATA3 staining in cytological
materials of prim
The aim of the study was to assess the diagnostic utility
of GATA3 immunocytochemistry in detecting the breast
origin of metastatic sites in cytological materials and to
compare GATA3 expression with those of conventional
and commonly used breast markers; mammaglobin and
Cytopathology reports from the Cytology archives of
the National Cancer Institute in Cairo, Egypt, during
the period between December 2013 and June 2015 were
searched retrospectively for the keywords “metastatic
breast carcinoma”. One hundred and thirty three cases
were recognized. They included seventy seven fine needle
aspiration cytology (FNAC) materials and fifty six exfoliated
serous effusion samples. Inclusion criteria included: a) cases
which had cytological reports of metastatic carcinoma, b)
cases which had previous documented histories of primary
breast carcinoma that were confirmed either by prior
histopathological diagnosis of excised breast lump or prior
cytopathological diagnosis of aspirated primary breast mass
and c) absence of any other detectable primary tumors on
routine metastatic follow up, d) availability of adequate
formalin-fixed, paraffin-embedded cell block materials
for ICC study. As a control group, 45 cytological materials
from well recognized non mammary metastatic tumors
were also analyzed for GATA3 staining. A minimum of
four Papanicolaou stained slides and a cell block section
were prepared for each case. All included archival slides
were reviewed to confirm the diagnosis and to assess
adequacy of cell blocks in order to use ICC. The presence
of at least 5 groups with at least 5 metastatic tumor cells
within each group was considered to be adequate cell block
materials and was included in the current study 4
Immunocytochemical Staining and Assessment
A 4-μm section was cut from each paraffin-embedded cell
block and mounted onto positively charged slides. The slides
were subjected to the ICC technique using a streptavidinbiotin-
peroxidase according to the manufacturer’s protocol.
The immunostaining was accomplished with BenchMark
XT automated slide stainer (a product of Ventana Medical
Systems). Sections were deparaffinized, rehydrated and
treated with 0.3% H2O2 for 5 min to block endogenous
peroxidase activity. They were then exposed to heatinduced
antigen retrieval. Antibody against GATA3 (mouse
monoclonal antibody, clone (L50-823), CELL MARQUE, Ventana Medical System was used. Diaminobenzidine
was used as a chromogen and Mayers hematoxylin as a
counterstain. Materials of metastatic breast carcinoma
were also assessed for mammaglobin staining using rabbit
monoclonal antibody, clone (31A5) as well as GCDFP-15
using rabbit monoclonal antibody, clone EP1582Y; CELL
MARQUE, Ventana Medical System. Appropriate positive
and negative control slides were prepared. Negative control
for all immunostaining was prepared by substituting the
primary antibodies with Phosphate Buffered Saline (PBS).
Positive staining control for GATA3 included sections
of urothelial carcinoma. Positive staining controls for
mammoglobin and GCDFP-15 included sections of breast
carcinoma known to be positive to these markers. Only
nuclear staining for GATA3 was scored, while cytoplasmic
staining for mammaglobin and GCDFP-15 was reported.
For GATA3, mammaglobin and GCDFP-15, staining
intensity and percentage of stained cells were reported.
Staining intensity was scored as 0 (no staining), 1+ (weak),
2+ (moderate), or 3+ (strong). Immunostained slides were
also assessed with respect to the percentage of stained
cells (0, no stained cells; 1+, 1%-10% of cells were stained;
2+, 11%-50% of cells were stained; 3+, >50% of cells were
stained). The final score was calculated by adding the
percentage of positive cells to intensity; a total score more
than 2 was considered as a positive staining result, and a
combined immunoreactivity score less than or equal to 2
was considered a negative result 8.
Data was analyzed using GraphPad Prism version 7.00 for
Windows (GraphPad Software, La Jolla, CA, USA: www.
Graphpad.com). Statistical significance was determined
by Fisher’s exact and Chi-square analysis for categorical
variables and ANOVA for numerical variables. The level
of significance was set at 0.05 or less. In addition, the
sensitivity, specificity, Positive predictive value (PPV),
Negative predictive value (NPV) and accuracy of the
marker were calculated.
|Clinicopathologic Characteristics of the Cases
One hundred and thirty three cytological materials of
metastatic breast carcinoma were assessed for positivity
of GATA3 immunostaining in cell block materials. All the
studied cases were female. The mean age was 44±10.3 with
range from 35 to 81 years; the median age was 55 years.
They included seventy-seven FNAC materials and fifty-six
exfoliated serous effusion samples. FNAC were aspirated
from the following anatomical metastatic sites: 42 from
lymph nodes (23 axillary, 16 cervical and 3 mediastinal), 18 from subcutaneous lump, 10 from lung lesions and 7 from
liver mass. Serous effusion materials included 40 pleural
and 16 ascitic effusions. Of 133 cases, 98 cases (73.7%) had
previous surgical resection of primary breast carcinomas and
documented histopathological reports. Histopathological
subtypes were ductal carcinoma (n=73), lobular carcinoma
(n=18), papillary carcinoma (n=3), mucinous carcinoma
(n=3) and metaplastic carcinoma (n=1). The remaining
35/133 cases had previous final cytopathological reports of
mammary carcinoma aspirated from primary breast lumps;
30 were ductal carcinoma, 4 lobular and one was papillary
carcinoma. As a control group, cytologic materials from 45
metastatic tumors from other non mammary defined sites were also analyzed for GATA3 staining. These materials
included 11 metastatic carcinomas from the female genital
tract, 10 from the thyroid gland, 9 of lung origin, 7 from the
gastrointestinal tract, 7 from the pancreas and one from the
GATA3 Immunocytochemical Staining on Metastatic
Positive GATA3 nuclear staining was detected in 82.7%
of metastatic breast carcinomas (110 of 133 cases). The
majority of stained cases, 85 cases (77.3%), had a score
of 6 or 5 (Figure 1-3). Twelve cases had a score of 4 while
thirteen cases had a score of 3 (Figure 4) (Table I).
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|Figure 1: GATA 3 immunocytochemical staining in cytological
cell block from metastatic papillary breast carcinomas in axillary
lymph nodes, score 6 (IHC; x200).
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|Figure 2: GATA 3 immunocytochemical staining in cytological
cell block from metastatic ductal breast carcinomas in pleural
fluid, score 6 (IHC; x400).
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|Figure 3: GATA 3 immunocytochemical staining in cytological
cell block from metastatic lobular breast carcinomas in cervical
lymph node, score 6 (IHC; x400).
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|Figure 4: GATA 3 immunocytochemical staining in cytological cell
block from metastatic ductal breast carcinomas in supraclavicular
lymph node, score 3 (IHC; x400).
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|Table I: GATA3 immunostaining scores among positive metastatic cases sorted by primary sites
Five out of 45 (11.1%) non mammary metastatic adenocarcinomas
were positive for GATA3; two positive cases
were metastatic carcinoma from lung to cervical lymph
nodes while two cases were metastatic uterine and ovarian
carcinoma in ascitic fluid and supraclavicular lymph nodes,
respectively. The remaining case was metastatic urothelial
carcinoma to subcutaneous tissue. They had scores of 3 and
5 (Table I). GATA3 was not detectable in any metastases
from thyroid carcinoma as well as gastrointestinal tract and
Sensitivity of GATA3 for identifying breast primary origin
in metastatic tumors was 82.7% (95% confidence interval
[CI]: 75.3% to 88.3%) while the specificity for absence of
breast differentiation in metastatic tumors was 88.9% (95%
CI: 76.1% to 95.65). Positive predictive value (PPV) was
95.7% (95% CI: 90% to 98.4%). Negative predictive value
(NPV) was 63.5% (95% CI: 51.1% to 74.3%). The overall
accuracy was 84.3% (95% CI: 78.2% to 88.9%) (Table II). A
statistical significant difference was demonstrated between
GATA3 staining in metastatic breast and non breast
carcinomas cases (p < 0.001). No GATA3 staining was
detected in benign cells set in the background (inflammatory
cells, mesothelial cells or stromal cells). No significant background staining was reported. GATA3 staining results
among different breast carcinoma histological subtypes
were reported in Table III. No statistical difference was
detected between GATA3 staining and different main
breast histological subtypes (p > 0.05).
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|Table II: Relation between GATA3 staining results and primary sites of metastases
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|Table III: GATA3 staining results among different breast carcinoma subtypes
GATA3 Immunocytochemical Staining Compared to
Mammaglobin and GCDFP-15 Expressions
Assessment of other conventional breast markers,
mammaglobin and GCDFP-15, were carried out on
metastatic breast carcinoma cell blocks to compare their
results with that of GATA3. Mammaglobin staining was
positive in 94 out of 133 cases (70.7%). GATA3 staining
for metastatic breast carcinoma was significantly higher
compared with mammaglobin staining (p = 0.03).
Mammaglobin staining tended to be less diffuse and less
intense than GATA3; forty-six out of 94 mammaglobin
positive metastatic breast carcinomas (48.9%) demonstrated
scores of 6 and 5 (Figure 5) (Table IV). The difference
was significant (p < 0.001). GATA3 and mammaglobin
values were concordant in 93 of 133 cases (69.9%); 82
cases were GATA3 positive / mammaglobin positive and
11 cases were GATA3 negative / mammaglobin negative.
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|Table IV: Distribution of immunocytochemical scores of GATA3, mammaglobin and GCDFP-15 among positive metastatic breast cases
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|Figure 5: Mammaglobin immunocytochemical staining of
metastatic breast carcinoma cell block in subcutaneous nodule,
score 5 (IHC; x400).
Discordant results were found in 40 of 133 cases (30.1%). Of
these, GATA3 was positive and mammaglobin was negative
in 28 cases. GATA3 was negative and mammaglobin was
positive in 12 cases. Adding mammaglobin to GATA3
resulted in improvement in GATA3 sensitivity to reach
91.7% for identifying the breast primary.
After staining of GATA3 and mammaglobin, 51 of 133
metastatic breast carcinoma cell blocks had sufficient
material for further evaluation of GCDFP-15. Among
these 51 cases, GATA3 expression was detected in 40
(78.3%). On the other hand, 24 cases (47.1%) were positive
for GCDFP-15 with background staining in 9 cases. All
GCDFP-15 positive cases were positive for GATA3. Sixteen
cases were GATA3 positive but GCDFP-15 negative.
The remaining 11 cases were negative for both markers.
GATA3 staining for metastatic breast carcinoma was
significantly higher compared with GCDFP-15 staining (p
< 0.001). Diffuse and intense staining was detected in only
8 cases (33.3%) of GCDFP-15 positive metastatic breast
carcinomas (Table IV). Adding GCDFP-15 to GATA3
resulted in no improvement in GATA3 sensitivity.
Although a metastatic tumor is an advanced stage cancer
and considered fatal, proper treatment can relieve tumorrelated
symptoms, delay cancer progression, prolong life
and improve the quality of life 9
. It makes a big therapeutic
and outcome difference when the primary organ of
metastasis is well known 10
. Identification of breast
differentiation in metastatic sites, based on morphology, is
a diagnostic challenge. The most commonly used markers,
mammaglobin and GCDFP-15, tend to be specific but have
low sensitivity and can be difficult to interpret in small
. GATA3 is a promising marker for breast
differentiation. Although GATA3 expression has been well
studied in histological specimens, the use of GATA3 in
cytological materials is understudied 11
. The main focus
of the current study was to evaluate GATA3 as a diagnostic
marker for metastatic breast carcinoma in cytological
In the current work, positive GATA3 nuclear staining
was detected in 82.7% of metastatic breast carcinomas
(110/133). In the literature, the GATA3 positivity rate
has been reported to range from 75 to 100% 12-14. The
reasons of this wide range might be related to the number
of studied cases or sample preparation (cell block or direct
smear). Leng et al., in their 2017 work reported GATA3
positivity in 71% of cell block sections and 89% of smear
samples 13. They reported that cell block materials are
more reliable as optimal samples for immunostaining
in cytological materials because fixation and staining
procedures are similar to that used for histologic samples.
The differences in GATA3 expression rates between the
published studies might have also resulted from using
different antibody clones or scoring systems 15. Different
sensitivities of using different clones on breast resection
specimens were reported in previous reports 16,17. Other
contributing factors for expression variation could also
be related to tumor characteristics (grade and molecular
subtypes) or technical causes (antigen retrieval methods,
dilutions or incubation times) 8. Furthermore, in the present study, GATA3 expression demonstrated a diffuse
and intense staining pattern (score 6 and 5) in 77.3% of
positive cases, suggesting that GATA3 expression was
evenly distributed with minimal staining variation within
the tumor. Therefore GATA3 can reliably highlight tumor
cells in samples with dispersed cells among a normal or
reactive background. Our result was in accordance with
previous reports 4,8.
On the other hand, GATA3 staining demonstrated
relatively high specificity (88.9%) in the present work. Five
out of 45 (11.1%) non mammary metastatic carcinomas
were positive for GATA3. A statistically significant
difference was demonstrated between GATA3 staining
in metastatic breast and non breast carcinoma cases (p <
0.001). These findings agreed with others who reported
that a small but significant percentage of metastatic non
mammary, non urothelial carcinoma can express GATA3
8,13. Thus they recommended using the GATA3 marker
as a part of panels to exclude or confirm other non breast
origin. Others reported that all the studied non breast, non
urothelial metastatic carcinomas were negative 9,15. In
our study, 2/9 (22.2%) of metastatic carcinoma from the
lung and 2/11 (18.2%) of metastatic female genital tract
carcinoma were GATA3 positive. These percentages were
higher than that reported in the literature on surgical
specimens 18. This might be due to small numbers of
cases in the current work and difference in materials used.
Because metastatic adenocarcinomas from the lung and
female genital tract have cytomorphological overlapping
features with metastatic breast carcinoma, cytopathologists
should be aware of GATA3 staining in such tumors to
In the present study, no statistical difference was detected
between GATA3 staining and various main breast
histological subtypes (p >0.05). This result was in keeping
with that reported by others 12,15. In the present work, no
GATA3 expression was detected in the benign cells set in
the background. Some authors observed weak positivity in
a minority of benign lymphoid cells in some cases 4,9. In
surgical specimens, positivity in a considerable percentage
of reactive and malignant mesothelial cells was observed
18. In other studies, no GATA3 expression was detected
in such cells 9,13.
Another aim of the current work was to compare the
positivity of GATA3 with that of mammaglobin and
GCDFP-15. In our study, GATA3 staining revealed superior
sensitivity compared to the sensitivities of mammaglobin
and GCDFP-15, the difference was statistically significant
(p = 0.03 and p < 0.001, respectively). These results were in accordance with previously published studies 4,8,15. If the
three markers were used in a panel, adding mammaglobin
to GATA3 markers resulted in improvement of GATA3
sensitivity (91.7%) as it could identify 12 additional
cases that were GATA3 negative. Adding GCDFP-15 to
GATA3 showed no sensitivity improvement. Therefore,
GATA3 could be used as a panel with the mammaglobin
marker for breast differentiation. Others have agreed with
these results 9,19. Based on experience with cytological
materials, markers with nuclear staining (such as GATA3)
are superior to those with cytoplasmic staining such as
mammaglobin and GCDFP-15 1,13. Less background
staining of GATA3 adds new advantages over the other two
In conclusion, GATA3 is a sensitive marker for diagnosing
metastatic breast carcinoma in cytological cell block
materials. GATA3 is more sensitive than mammaglobin
and GCDFP-15 with more diffuse and stronger expression.
Adding mammaglobin to GATA3 resulted in improvement
of the sensitivity. GATA3 was occasionally positive in
some metastatic non mammary carcinomas that might
be included in breast differential diagnosis; sole GATA3
staining should therefore be interpreted in conjunction
with morphological, clinical, radiological findings to avoid
a misdiagnosis when working with a tumor of unknown
origin or used as part of an immunostaining panel.
However, it is recommended to use GATA3 in a large
number of non mammary metastatic tumors on cytological
materials to detect its exact incidence in these tumors.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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