2014, Volume 30, Number 2, Page(s)
Lymphatic Vascularization in Primary Breast Cancer: HER2 Overexpressing Tumors Contain More Lymphatics than Steroid Receptor Positive, Triple-Positive and Triple Negative Breast Carcinomas
Savelina POPOVSKA, Ivan IVANOV
Departments of Pathology, Medical University-Pleven, PLEVEN, BULGARIA
Keywords: Breast neoplasms, Estrogen receptor, Progesterone receptor, HER2, Lymphangiogenesis
The aim of this study was to examine the relationship
between the immunohistochemical subtypes of invasive breast cancer
and lymphatic vascularization.
Material and Method: One hundred and seventy nine cases of
randomly selected invasive breast cancer patients, surgically treated
between 2004 and 2007, were retrospectively studied. These were
classified into steroid receptor positive (steroid receptor positive/
HER2 negative), triple positive (steroid receptor and HER2
positive), triple negative (steroid receptor and HER2 negative) and
HER2 overexpressing (steroid receptor negative /HER2 positive)
carcinomas. Appropriate immunostaining and in-situ hybridization
techniques were applied and results were statistically analyzed.
Results: The median intra-tumor lymphatic vascular density and the
median intra-tumor relative lymphatic vascular area were found to
differ significantly among the studied groups of breast cancer (KW
=49.8611; p<0.0001 and KW =21.5122; p=0.0001 respectively). There
was no significant difference in the incidence rate of axillary node
involvement among the studied groups of breast cancer (χ2=1.66;
Conclusion: The present study indicates that HER2 overexpressing
breast carcinomas have a consistent increase of intra-tumor lymphatic
vessel counts as compared to all other subtypes. It is suggested that
the newly formed vessels are probably not the only essential factor
for lymphogenic spread of HER2 overexpressing breast carcinomas
as they are not related to an increased incidence of lymph node
metastases compared to the other studied subgroups.
There are several classifications of breast carcinomas based
on their immunohistochemical expression profile for
estrogen receptor (ER)/ progesterone receptor (PgR), HER2,
epidermal growth factor receptor (EGFR), cytokeratin
(CK) 5/6 and Ki-671-6
. Accordingly, breast carcinomas
(BC) are divided into subtypes which are believed to be
relevant to prognosis2,5,7,8
. Both triple negative
and HER2 overexpressing / ER negative carcinomas are
characterized by poor prognosis9
. Potential factors that
may contribute to the unfavorable prognosis of HER2
overexpressing tumors are increased angiogenesis10,11
Raica et al. have reported high levels of expression of vascular
endothelial growth factor (VEGF)-C/vascular endothelial
growth factor receptor (VEGFR)-3 and D2-40 in HER2 and
luminal B types, and low rates of expression in basal-like type.
In addition, the same authors have found the lowest value
of both intratumor and peritumor lymphatic microvessel
density (LV D) in normal-like type of BC14. Presently, it
is not clear whether some types of BCs display a consistent
increase of lymphatic vascularization (LV ). In addition there
is no evidence that LV D is associated with an increase in the
metastatic potential via the lymphatic pathway.
The purpose of this study was to find any relation between
different subtypes of invasive breast cancer, subdivided
according to their steroid receptor (ER, PgR) and HER2
status, and lymphatic vascularization present within or
around the primary tumor, using immunohistochemistry.
One hundred and seventy nine randomly selected
cases of invasive BC, surgically treated between 2004 and
2007 in the University Hospital in Pleven, Bulgaria, were
retrospectively studied. Archival formalin fixed paraffin
embedded tissues as well as pathology reports and casehistory charts were retrieved and analyzed. All patients
were female, ranging in age between 30.0 and 81.0 years
(average 60.4 yrs).
The 179 cases studied included 147 (82.12 %) invasive ductal
carcinomas, 26 (14.52 %) invasive lobular carcinomas, 3
(1.68 %) mucinous carcinomas, and 3 (1.68 %) medullary
carcinomas. Tumors were staged according to the AJCC15 and graded according to the Elston & Ellis criteria16.
Immunohistochemistry: All cases were immunostained for
ER, PgR, HER2 and D2-40. Details concerning the primary
antibodies used are presented in Table I. All immunostains
were manually processed. The FLEX EnVISION (DAKO)
method was used for immunohistochemical staining.
Immunohistochemical reactions were developed with 3-3’
diaminobenzidine and sections were counterstained with
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|Table I: General information concerning the used primary antibodies: clone, working concentration, manufacturer and antigen
retrieval method used
Evaluation of staining: Manual readings were carried
out independently by the two investigators (S.P. and I.I.).
Discordances were discussed until a consensus was reached.
HER2 was accepted as positive if immunohistochemistry
(IHC) reaction for HER2 was complete, uniform, and
presented intense membrane staining of > 30% of invasive
tumor cells (3+), or if there were more than six HER2 gene
copies per nucleus in >50% of the cancer cells on CISH. If
IHC staining for HER2 was complete, uniform membrane,
with moderate intensity of >30% of invasive tumor cells result
was considered equivocal – (HER2 - 2+), and additional
testing with CISH was performed. Cases with incomplete,
weak (1+) or missing (0) immunostaining on IHC, as well as
cases with less than five HER2 gene copies per nucleus, were
considered negative9,17. ER and PgR were considered
positive if >10% of invasive tumor cells displayed weak,
moderate or strong immunostaining18. Vessels were
interpreted as lymphatic structures if most of the lining cells
showed intermediate to strong cytoplasm immunostaining
for D2-40 and lumina were devoid of red blood cells.
CISH: CISH was performed on 4 μm tissue sections. Only
cases with HER2 2+ receptor status were retested with
the CISH method. The tissue sections were treated in
accordance with the protocol provided by the manufacturer
(CISH™ Tissue Pretreatment Kit, Zymed). The CISH signals
were visualized and assessed using a bright-field microscope
at magnification 10 x/ 60 x objectives.
IHC classification of breast tumors based on ER, PgR
and HER2: IHC markers and CISH were used to define the
breast cancer subtypes which were categorized as: Steroid
receptor positive (SRP) - (ER+ and/or PR+, HER2 - ),
Triple positive tumors (TPT) (ER+ and /or PR+, HER2+),
HER2 overexpressing (HER2+, ER- and/or PR-) and Triplenegative
tumors (TNT) (ER-,PR- and HER2-), modified
from the one suggested by Onitilo AA et al.9.
Definition of LV I : Lymphatic vascular invasion (LV I) was
assessed on D2-40 stained slides. Any tumor-cell clusters
present within D2-40 positive vessels were accepted as LV I.
Lymphatic vascularity assessment: The number of
lymphatic vessels was assessed by the two investigators.
D2-40 stained slides were covered by a grid 20x20mm,
composed of 25 small squares 4x4mm each (Figure 1A).
These slides were then scanned at low magnification 40x
(4x objective 10x eye-peace). The LV count was assessed
in each square and the result was filled in a specially
developed individual hot-spot identification table (Figure
1B). If differences in vascular counts were overestimated by
+/- 10 %, counting was repeated.
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|Figure 1: A) An example
of covering glass stamped
with 20x 20mm grid.
Any grid printed on
transparent material (plate)
can be mounted instead
B) Individual hot-spot
identification table – an
example of the hot-spot
identification table used in
After hot spots had been identified, the highest possible
intratumor (inside the tumor and its stroma) and peritumor
(at the tumor front of invasion and around the tumor)
lymphatic vascular density (LV D) was estimated by the two investigators at magnification 100x (10x objective 10x eyepeace).
If differences in vascular counts were overestimated
by +/- 10 %, the counting was repeated.
Areas with the highest vascularization were photographed
using an Olympus BX 40 microscope, equipped with an
Olympus digital camera 5050 zoom and digital images
were saved. A computerized image analysis was performed
using Image Tool software. After spatial calibration of the
software, images were used for the calculation of intra- and
peritumor relative lymphatic vascular area (RLV A). RLV A
was calculated as the area occupied by lymphatic vessels as
related to the whole area of the field (1.2mm2). For optimal
results, measurement was performed manually.
Statistical analysis: Statistical analysis was performed with
the Statgraphics plus 2.0 software package. Comparison of
all parametric variables was statistically studied as it follows:
initially, normality of the studied variables was tested with
Kolmogorov – Smirnov test. Since all parametric variables
were not with normal distribution, the Kruskal–Wallis
test was used for comparing lymphatic vascularization
parameters. The relation of the IHC determined subtypes
of breast cancer to lymphatic vascularization parameters
was initially tested with the Kruskal–Wallis test. After
analyzing lymphatic vascularization parameters, a posthoc
analysis test was carried out by applying the Mann–
Whitney (Wilcoxon) W two-tailed test (Statgraphics plus
2.0 software package) to identify the presence of significant
differences between the lymphatic vascularization observed
in the studied subgroups of breast cancer.
The relation of axillary node status to lymphatic
vascularization parameters was tested using Kruskal–Wallis
test. Differences in non-parametric variables were tested using the Chi–square test. Probability values of p<0.05
were considered to represent a significant difference for the
Kruskal–Wallis test and Chi–square test and probability
values of p<0.0083 for the Mann–Whitney (Wilcoxon) W,
after Bonferroni correction was applied.
Intratumor lymphatic vessels were mostly present in the
tumor margins of BC showing infiltrative growth pattern
in SRP, TPT and HER 2 subtypes (Figure 2A,B
with predominantly pushing growth (some of the TNT)
had scanty intratumor lymphatic vessels (Figure 2C
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|Figure 2: A) Steroid receptor positive breast carcinoma – a
predominantly infitrative growth pattern was observed. Co-opted
terminal ducto-lobular unit (arrow head) as well as lymphatic vessels
that looked collapsed (arrows) were observed in the periphery of
the tumor. The lymphatic vessels in the periphery of the tumor
were generally preserved, almost similar in size and morphology to
normal lymphatic vessels (contour arrows) (D2-40; x100).
B) HER2 overexpressing breast carcinoma - infiltrative growth
pattern was observed. Lymphatic vessels were seen in the tumors’
periphery (arrows). Most of those vessels seemed somewhat smaller
than normal and collapsed (D2-40; x100).
C)Triple-negative breast cancer – presented with typical “pushing
margin” growth pattern. The tumor had no intratumor lymphatic
vessels; peritumor lymphatic vessels are designated with (contour
arrows) (D2-40; x100).
Intratumor lymphatic vessels appeared predominantly as
vascular clefts, while peritumor lymphatic vessels were
characterized by well-evident to dilated structures. In 48
(26.82%) of the cases, lymphatic vessels were harboring tumor cell emboli. The median intratumor lymphatic
vascular density (ILV D) was found to be significantly
different among the studied subtypes of breast cancer,
subdivided on the basis of IHC (KW =49.8611; p<0.0001)
(Figure 3A). The median value for ILV D in HER2
overexpressing carcinomas was 6.5 lymphatic vessels, and
was significantly higher when compared with a median
of 0.00 (no intratumor lymphatic vessels) in the other BC
subtypes (SRP; TPT and TNT). No significant difference
was found between SRP- TPT, SRP - TNT and TPT – TNT.
Details are presented on Table II.
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|Figure 3: Quantitative comparison of lymphatic vascularization of different molecular subtypes of breast cancer divided on the basis of IHC.
A) Intratumor lymphatic vascular density (ILV D), B) Intratumor relative lymphatic vascular area (IRLV A), C) Peritumor lymphatic
vascular density (PLV D), D) Peritumor relative lymphatic vascular area (PRLV A).
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|Table II: Comparison between the lymphatic vascularization observed in the studied subgroups of breast cancer, divided on the basis
of IHC profile for ER, PgR and HER2
The median intratumor relative lymphatic vascular area
(IRLV A) was found to be different in the subtypes of BC,
divided on the basis of IHC (KW =21.5122; p=0.0001) (Figüre 3B). The median value for IRLV A in HER2 overexpressing
carcinomas was 0.0037316, and was significantly increased compared to the median values of IRLV A 0.00 (no intratumor
lymphatic vessels) in SRP and TPT subtypes. No significant
difference was found between HER2 overexpressing tumors
and TNT as well as between SRP- TPT, SRP- TNT and TPT–
TNT. Details are presented on Table II.
Some not statistically significant differences in the median
peritumor lymphatic vascular density (PLV D) were found
among the different molecular subtypes of breast cancer
considered (KW =2.11619; p=0.54864) (Figure 3C). No
differences were found in the median values for PLV D
when the different subgroups of BC were compared. Details
are presented on Table II.
In general, no significant differences were found in the
median peritumor relative lymphatic vascular area (PRLV A)
among the subtypes of breast cancer divided on the basis of
IHC (KW =4.89442; p=0.179691) (Figure 3D). From all the
analyzed groups only TPT and HER2 were found to differ significantly in their median values of PRLV A. Details are
presented on Table II.
The distribution of tumor size in the studied groups is
presented on Table III. Steroid receptor positive tumors
and TPT’s were likely to be predominantly low grade
compared with HER2 overexpressing tumors and triplenegative
BC that were predominantly high grade lesions.
The distribution of the studied cases by grade and subtypes
of breast cancer, divided on the basis of IHC, is presented
on Table III. The age of initial clinical presentation was
different in the different breast cancer subtypes (Table III).
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|Table III: Correlation between some of the main predictive factors in breast cancer and immunophenotype
No correlation was found between lymph node status
and the immunophenotype of the tumor (Table III).
There was no significant difference in the incidence of
lymphatic vascular invasion found in tumors with different
immunophenotypes (Table III).
Analysis of the relationship between intra- and
peritumoral lymphatic vascularization and lymph node
status demonstrated that both indicators of intratumor
vascularization (IRLV A and ILV D) were increased in node
positive patients compared to node negative patients.
At the same time, both PRLV A and PLV D were not
associated with the axillary lymph node status. Details are
presented on Table IV.
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|Table IV: Lymphatic vascularization parameters and their relation to axillary lymph node status
The controversy concerning the presence of
lymphangiogenesis in breast cancer is open to questions12,19-24
. Breast carcinomas might be divided into
or molecular subtypes1-6
for the latter are not widely accepted, and several variations
in definitions of molecular subtypes of breast carcinomas
Controversies also exist for the score of estrogen receptors
(ER). According to ASCO/CAP guidelines, positivity for
hormone receptors in breast cancer is considered when
more than one percent of the total tumor cell proliferation
expresses ER and/or PgR receptors. This threshold for
positivity proved useful in clinical practice because of
its significance in clinical prognosis and prediction25.
Nevertheless, other authors view the cut-off point of ER/
PgR positivity of 10 % as the minimum percentage of
immunopositive tumor cells required to consider hormone
receptor positive status18. Despite the fact that the 10%
positivity threshold for steroid receptors in BC is an old
concept in terms of treatment and survival, we believe it
is the positivity threshold that has significant meaning for
angiogenesis and potentially for lymphangiogenesis as well.
As Elkin M et al. mentioned, 7%-17% is the ER expression in
the normal breast epithelium and probably more ER positive
tumor cell are needed to induce angiogenesis in BC26.
The present study confirms the data of the increased
lymphatic vascularity associated with HER2 over-expression
in the HER2 subtype14. Accordingly, increased median
values of ILV D and IRLV A in invasive HER2 overexpressing
but not in all HER2 pathway-driven breast carcinomas
(TPT) have been demonstrated. Surprisingly, IRLV A in
invasive HER2 tumors was not significantly different from
IRLV A in invasive TNT. We believe that this is due to the
relatively small area occupied by the greater in number but
collapsed intratumor lymphatic vessels.
Increased lymphatic vascularization was seen mostly at
the periphery of invasive HER2 tumors a finding that
might be interpreted as the result from entrapment of pre-existing vessels by the invading tumor. In such cases,
entrapped lymphatic vascularization by the tumor ought
to be of the same magnitude as the vessels located within
the surrounding unaffected tissue. This was not the case
as peritumor lymphatic vascularization (vascularization of
morphologically normal breast tissues) was found similar
to all the other tumor types which on the other hand
had no increased intratumor lymphatic vascularisation.
This would indicate that HER2 overexpressing breast
carcinomas do have denser lymphovascularization caused
by active lymphangiogenesis inside the tumor. Additionally,
peritumor lymphatic vascularization parameter PRLV A
was found to be significantly increased in HER2 tumors
compared to TPT, but since it was not supported by
increased peritumor vascular counts, this was accepted as
significant dilation of the peritumor vessels.
The present study demonstrates that the patients with
positive axillary node status have significantly increased
ILV D and IRLV A, compared to patients with negative
axillary lymph node status.
Different staining procedures, lack of uniform definition of
peri- and intratumor lymphatic vessels and the wide variety
of studied breast carcinomas contribute to the presence of
serious discrepancies concerning the role of intratumor
lymphatic vessels in breast cancer (reviewed in27).
One article that defined intra- and peritumor vessels in
BC in accordance with our understanding demonstrated
a “significantly higher maximal perimeter of intratumoral
and peritumoral lymph vessels” in node positive breast
cancer patients. At the same time, lymphatic vascular area
and the number of lymphatic vessels were not associated
with lymph node status21. We believe that the present
discrepancy with our result might be caused by the fact that
the abovementioned study concerned only inflammatory
Results, suggesting the important role of intratumor
lymphatic vessels for lymphogenic spread of primary tumors
were observed in early gastric cancer28. In spite of higher
lymphangiogenesis in HER2 carcinomas, no statistical
difference was present for lymph node metastases with the
other types of carcinomas not showing angiogenesis and
more specifically axillary node metastases were present in
half of the HER2 carcinomas, while axillary nodes were
affected in more than half of the cases of SRP, TPT and TNT
that were closely matched by tumor size and grade.
An apparent paradox was that even the better differentiated
carcinomas with significantly lower median lymphatic
vascularity such as tumors from the SRP group had metastasized to the axillary lymph nodes more frequently
than the HER2 overexpressing carcinomas that were
moderately to poorly differentiated neoplasms with
relatively higher median lymphatic vascularity. All these
data point to the fact that intratumor neoformed lymphatics
are of limited functional capacities for generating metastases
while preexisting vessels outside the tumor burden are more
apt to route tumor emboli.
In conclusion, the present study indicates that HER2
overexpressing tumors have increased intratumor
lymphatic vascularization as compared to all other subtypes
of breast carcinomas. However, this increase appears to be
of low clinical impact since the metastatic rate of HER2
overexpressing breast carcinomas is not higher than that
of any other subtype of breast cancer, and probably reflects
the fact that the newly formed intratumor lymphatic vessels
in invasive HER2 overexpressing breast carcinoma are not
We would like to thank to Vincenzo Eusebi who gave us
scientific guidance, and encouraged a clear presentation of
our scientific results.
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