Journal of Cancer Therapy, 2012, 3, 814-821
http://dx.doi.org/10.4236/jct.2012.325103 Published Online October 2012 (http://www.SciRP.org/journal/jct)
Lymphangiogenesis as a Prognostic Marker in Breast
Cancer Using D2-40 as Lymphatic Endothelial Marker—A
Preliminary Study*
Mumtaz A. Ansari1, Vaibhav Pandey1, Vivek Srivastava1, Mohan Kumar2, R. N. Mishra3,
Anand Kumar1#
1Department of General Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India; 2Department of Pathol-
ogy, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India; 3Department of Biostatistics, Institute of Medical
Sciences, Banaras Hindu University, Varanasi, India.
[e-mail], #
Received August 23rd, 2012; revised September 26th, 2012; accepted October 5th, 2012
ABSTRACT
We studied tumour lymphangiogenesis and lymphatic invasion using D2-40 endothelial marker in 35 breast cancer pa-
tients treated by primary surgery and correlated it with various clinico-pathological prognostic parameters. Lymphan-
giogenesis was quantified using lymphatic micro vessel density (LMVD) by counting the immunostained lymphatic
microvessels at 200×. The mean age was 45.97 ± 12.09 years (range 30 - 80 years). LMVD ranged from 5/hpf to 56/hpf
with a mean score of 13.4 ± 10.8 and median of 9. The median value of 9 was taken to classify patients into a low or
high LMVD. LMVD correlated significantly with tumour size (p = 0.003), histological grade (p = 0.046), lymph node
status (p = 0.030). There was no significant correlation of LMVD with stage, estrogen receptor, progesterone receptor
or HER2/neu immunoreactivity. Lymphovascular invasion on D2-40 staining [LVI-D40] was found in 13 (37.1%) cases
compared to 6 cases (17.1%) on H & E staining showing a poor agreement (k = 0.244). LVI correlated significantly with
lymph node status (p = 0.011). There was a strong association between tumour size (p = 0.142), histological grade (p =
0.066) though the correlation was not statistically significant. No correlation was found with stage, estrogen receptor,
progesterone receptor or HER2/neu immunoreactivity. The mean LMVD in LVI positive patients was higher (22.85 ±
13.29) as compared to LVI negative patients (7.95 ± 2.05) and this was statistically significant (p = 0.001). Increased
D2-40 detected LMVD and LVI correlated with poor prognostic parameters.
Keywords: Breast Cancer; Lymphangiogenesis, D2-40; Prognosis
1. Introduction
Breast cancer is the commonest cancer affecting women
worldwide. Currently its management involves the as-
sessment of various predictive and prognostic markers.
Lymph node (LN) status is the most important inde-
pendent clinical prognostic factor for patients with breast
cancer [1]. This important prognostic benefit is not found
in node negative patients and hence there is a need of a
marker which can predict the subset of patients who will
develop lymph node involvement eventually and can also
serve as a prognostic marker. Lymphangiogenesis refers
to the development and proliferation of new lymphatics
from host vessels. Quantification of lymphangiogenesis
has been done by measuring the lymphatic microvessel
density (LMVD). Changes in environment and composi-
tion of lymphangiogenic extracellular matrix, which is
actively involved in tumor cell chemotaxis, may affect
the function of both preexisting and newly formed lym-
phatics, promoting tumor cell invasion and dissemination.
It has also been proposed that entry of cancer cells into
the lymphatic vasculature might be facilitated by the
higher permeability of proliferated lymphatic vessel and
this enhances lymphatic metastasis assuming that tumor
cells are passively taken up by lymphatic vessels along
with the protein-rich interstitial fluid.
Lymphangiogenesis and invasion of lymphatics by
tumour cells can serve as a surrogate marker in such
situations. Though recent research on angiogenesis in
cancer kinetics has been widely studied, yet lymphan-
giogenesis and the process of lymphatic invasion fol-
lowed by metastasis to regional lymph nodes remains
*Conflict of interest: the authors of this article declare no conflict of in-
terest.
#Corresponding author.

Lymphangiogenesis as a Prognostic Marker in Breast Cancer Using D2-40 as Lymphatic Endothelial
Marker—A Preliminary Study
815
poorly understood. This lacuna in knowledge is primarily
because of lack of lymphatic endothelial markers that can
specifically identify the lymphatic endothelium.
Studies have demonstrated the clinical usefulness of
quantifying lymphangiogenesis by lymphatic microves-
sel density (LMVD) estimation in head & neck and lung
cancer using specific markers for lymphatic vascular
endothelium [2,3]. Since then, many studies have evalu-
ated the LMVD as a prognostic factor for breast cancer
[4,5]. LMVD was found to significantly correlate with
the presence of regional or distant metastases and further
shown to have a prognostic significance for breast cancer.
However there was failure of consistent correlation be-
tween the LMVD and clinical outcome in breast cancer
[5]. Controversies also exists about the role of peritu-
moural versus intratumoural lymphatics, site of lym-
phangiogenesis and the significance of mere lymphatic
proliferation to qualify as an indicator for future nodal
involvement. The lack of sensitivity of detecting only
lymphatic endothelium was the major draw backs in
these studies.
The monoclonal antibody D2-40 is shown in breast
and tonsillar tissue to selectively detect lymphatic vessels
in sections of both frozen and formalin fixed paraffin
embedded normal and neoplastic tissues [5]. The meth-
odology adopted to study lymphangiogenesis has been to
find out the LMVD and the presence of tumour emboli
within peritumoural endothelial lined spaces, defined as
lymphovascular invasion (LVI). The identification of
LMVD and LVI may permit the determination of pa-
tients at increased risk for axillary involvement and me-
tastasis and thus can serve as a prognostic marker in node
negative breast cancers.
The aim of this study was to determine the LMVD and
LVI using D2-40 and their interrelationship with estab-
lished prognostic markers in breast cancer.
2. Material and Methods
A prospective study was conducted in a single surgical
unit of a University hospital from March 2008 to Febru-
ary 2010. All patients of carcinoma breast admitted in the
unit during study period for surgery as primary modality
of treatment were included in study. The patients who
received any prior treatment like, lumpectomy, neo-ad-
juvant chemotherapy or Radiotherapy were excluded.
Patients who had any kind of breast surgery in past or
who did not give consent were also excluded. The diag-
nosis was made by FNAC from breast lump after detailed
clinical evaluation. Patients underwent modified radical
mastectomy followed by detailed histological examina-
tion of specimen, receptor status evaluation and LMVD
and LVI estimation using D2-40 as primary antibody.
Adjuvant treatment in the form of chemotherapy and/or
radiotherapy was given if indicated to complete the ther-
apy. After completion of treatment patients were kept on
three monthly follow up for two year and thereafter every
six months. Metastasis is recorded during the follow up
period.
Histological examination was done after hematoxylin
and eosin (H & E) staining and tumour was graded ac-
cording to the Bloom Richardson Grading system. The
presence of tumour emboli within peritumoural endothe-
lial lined spaces, stained with H & E was defined as posi-
tive LVI. Hormone receptor status was determined by
immunohistochemistry.
Blocks of the viable and tumour representative area
were selected for LMVD and LVI estimation using im-
munohistochemistry. Tissue sections (4μm thick) were
dewaxed and antigen retrieval was done by incubation in
microwave using citrate buffer (Ph 6) at 95˚C and 97˚C
for 10 minutes each. Then the sections were incubated in
3% H2O2 solution for blocking endogenous peroxidase.
Following this the tissue sections were incubated in pri-
mary antibody D2-40 [1:50 dilution] (DAKO corporation,
Denmark) solution in humid chamber for 30 minutes at
room temperature. After washing, the sections were
treated with biotinylated secondary antibody followed by
avidin coupled to biotinylated peroxidase at room tem-
perature [LSAB kit] (DAKO corporation, Denmark).
Immunohistochemical reaction were developed with di-
amino benzidine dihydrochloride (DAB) chromogen per-
oxidise substrate and counterstained with Haematoxylin
and Eosin. A block of archival tonsillar tissue served as
positive control [6]. For negative control, a slide was
prepared from the same tissue block and a preimmune
serum was used instead of the primary antibody.
Lymphatic Microvessel Density (LMVD) and
Lymphatic Vascular Invasion (LVI) Assessement
The determination of microvessels was done using crite-
ria described by Weidner et al, 1991 [7]. Lymphatic ves-
sels were defined as the vessels which have endothelium
with immune-positivity to D2-40 and had a lumen. The
sections were initially scanned at low magnification
(40×), thereby finding the areas with the highest number
of lymphatic microvessels (the hot spots). LMVD was
then determined by counting all D2-40 immunostained
vessels at high magnification (200×) in three hot spots
(Figures 1 and 2). Determination of staining was strictly
confined to hot spots. Microvessel counts were done by
two independent observers, naive to the patient’s patho-
logic and clinical status. The mean value of microvessel
densities observed by both investigators in each patient
was entered into further calculations. In case of in-
ter-observer difference of more than 30% in microvessel
count, the respective slides were reinvestigated by both

Lymphangiogenesis as a Prognostic Marker in Breast Cancer Using D2-40 as Lymphatic Endothelial
Marker—A Preliminary Study
816
Figure 1. Invasive breast carcinoma showing positive im-
munostaining of lymph vessels with D2-40 (100×). No tu-
mour embolus seen.
Figure 2. Lymphovascular bundle stained with D2-40 with
positive staining of lymphatic endothelium (200×). The ad-
jacent blood vessel endothelium is not stained.
observers using a discussion microscope (evident in <5%
of cases).
LVI was recognized as tumour cell nests floating
within empty spaces, which were surrounded by thin,
spindle shaped endothelial cells. A lymph vessel that
showed positive staining of the endothelium for D2-40
which surrounded the tumour cell nests was diagnosed as
positive for LVI (Figure 3). The evaluation of LVI by
the hematoxilin and eosin stained sections was also
documented for the study. The D2-40-stained slide was
assessed for lymph vessel invasion without knowledge of
the LVI status based on the hematoxilin and eosin stain-
ing. Interpretation of immunohistochemical results were
made without knowledge of clinical stage or the status of
other prognostic variables. LMVD and LVI were corre-
lated with other prognostic parameters besides each
other.
Statistical analysis was done using SPSS software ver-
sion 14. The mean difference between two groups was
Figure 3. Positive staining of lymphatic endothelium with
D2-40 outlining a tumour embolus in the lumen (200×). This
tumour embolus could not be visualized on the H & E sec-
tion as it completely obliterates the lumen.
calculated by student t test for unpaired data and chi
squared test was done for significance of difference
among two proportions. Correlation among qualitative
data was done by Pearson correlation. Kaplan Meier
curve was used to visualize the survival rate and log rank
test was performed to compare the survival rates. The
level significance was considered at 5% cut-off point.
The significance level more than 5% written as p > 0.05
was taken as statistically insignificant.
3. Results
A total of thirty five female patients entered into study
during the study period. The mean age was 45.97 ± 12.09
years (range 30 - 80 years). Clinicopathologic parameters
of these patients are given in Table 1.
Lymphangiogenesis was quantified by microvessel
density, using D2-40 as markers for lymphatic endothe-
lium. In our study LMVD ranged from 5/hpf to 56/hpf
with mean LMVD of 13.4 ± 10.8 and median LMVD of
9. The median value of 9 was taken to classify patients
into a low or high LMVD score as a reference value. The
cases thus were divided based on low or high LMVD and
was compared with other prognostic markers viz. tumour
size, stage, number of lymph node involvement, histo-
logical grade and receptor status. The results are shown
in Table 2. LMVD correlated significantly with tumour
size (p = 0.003), histological grade (p = 0.046), lymph
node status (p = 0.030). There was no significant correla-
tion of LMVD with stage, estrogen receptor, progester-
one receptor or HER2/neu immunoreactivity.
Lymphovascular invasion on D2-40 staining [LVI-
D40] was found in 13 (37.1%) cases compared to 6 cases
(17.1%) on H & E staining. Out of 13 LVI positive
cases, 9 cases were positive for LVI on D2-40 only and

Lymphangiogenesis as a Prognostic Marker in Breast Cancer Using D2-40 as Lymphatic Endothelial
Marker—A Preliminary Study
817
Table 1. Clinicopathologic characteristics of thirty five pa-
tients.
Characteristics
Age
Mean (years)
Range (years)
45.97 ± 12.09
30 - 80
Menstrual status, No (%)
Premenopausal
Postmenopausal
18 (51.4)
17 (48.6)
Tumour size (cm)
T 1
T 2
T 3
-
18 (51.4)
17 (48.6)
Clinical N-status
N0
N1
N2a
N2b
11 (31.43)
14 (40.0)
10 (28.57)
-
Stage
IIa
IIb
IIIa
6 (17.1)
16 (45.7)
13 (37.1)
Grade
I
II
III
1 (2.9)
10 (28.6)
24 (68.6)
No. of involved Lymph Nodes
0
1 - 3
4 - 9
>10
14 (40.0)
7 (20.0)
5 (14.3)
9 (25.7)
ER
Positive
Negative
3 (13.63)
19 (86.36)
PR
Positive
Negative
3 (13.63)
19 (86.36)
HER2/neu
Positive
Negative
9 (40.90)
13 (59.09)
negative on H & E staining while only 2 cases were posi-
tive for LVI on H & E but negative on D2-40 staining.
Four cases were positive on both D2-40 and H & E. The
kappa score obtained in our study showed poor agree-
ment regarding the LVI when comparing D2-40 immu-
nostained and H & E stained sections (k = 0.244). Table 3
shows comparision of LVI on D2-40 staining correlated
with other prognostic parameters. LVI correlated signify-
cantly with lymph node status (p = 0.011). There was a
strong association between tumour size (p = 0.142), his-
tological grade (p = 0.066) though the correlation was
not statistically significant. No correlation was found
with stage, estrogen receptor, progesterone receptor or
HER2/neu immunoreactivity. The mean LMVD in LVI
positive patients was higher (22.85 ± 13.29) as compared
to LVI negative patients (7.95 ± 2.05) and this was statis-
tically significant (p = 0.001). Five out of thirty five pa-
tients developed metastasis during follow up, three in
liver and two in spine. Four out of these five patients
showed lympho vascular invasion while one was nega-
tive (p = 0.032).
4. Discussion
Breast cancer is the second leading cause of cancer
deaths among woman worldwide [8]. The concept of
tumour induced lymphangiogenesis has been met with
Table 2. Comparison of LMVD with other prognostic pa-
rameters.
LMVD
<9 ≥9
No. (%) No. (%)
p-value
Mean tumour size 4.20 ± 1.61 6.10 ± 1.83 0.003
Stage
IIA
IIB
IIIA
4 (26.7)
7 (46.7)
4 (26.7)
2 (10.0)
9 (45.0)
9 (45.0)
0.388
Positive lymph node
1 - 3
4 - 9
>10
3 (100)
-
-
4 (22.2)
5 (27.8)
9 (50.0)
0.030
Grade
I
II
III
1 (6.7)
7 (46.7)
7 (46.7)
-
3 (15.0)
17 (85.0)
0.046
ER
Positive
Negative
1 (12.5)
7 (87.5)
2 (14.3)
12 (85.7)
0.907
PR
Positive
Negative
1 (12.5)
7 (87.5)
2 (14.3)
12 (85.7)
0.907
HER
Positive
Negative
4 (50.0)
4 (50.0)
5 (35.7)
9 (64.3)
0.512

Lymphangiogenesis as a Prognostic Marker in Breast Cancer Using D2-40 as Lymphatic Endothelial
Marker—A Preliminary Study
818
Table 3. Comparison of LVI with other prognostic parame-
ters.
LVI D2-40
Positive Negative
No. (%) No. (%)
p-value
Mean Tumour Size 5.92 ± 1.55 4.91 ± 2.11 0.142
Stage
IIA
IIB
IIIA
2 (15.4)
7 (53.8)
4 (30.8)
4 (18.2)
9 (40.9)
9 (40.9)
0.755
Grade
I
II
III
-
1 (7.7)
12 (92.3)
1 (4.5)
9 (40.9)
12 (54.5)
0.066
Positive lymph node
1-3
4-9
>10
1 (8.3)
3 (25.0)
8 (66.7)
6 (66.7)
2 (22.2)
1 (11.1)
0.011
ER
Positive
Negative
1 (9.1)
10 (90.9)
2 (18.2)
9 (81.8)
0.534
PR
Positive
Negative
1 (9.1)
10 (90.9)
2 (18.2)
9 (81.8)
0.534
HER
Positive
Negative
4 (36.4)
7 (63.6)
5 (45.5)
6 (54.5)
0.665
Mean LMVD 22.85 ± 13.29 7.95 ± 2.05 0.001
Metastatic disease in
follow up
Present
Absent
4 (30.8)
9 (69.2)
1 (4.5)
21 (95.5)
0.032
skepticism in the past and some what neglected. It was
considered that tumour associated lymphatics were pre
existing and not newly formed [9]. Recent research has
provided sufficient evidence that tumours are not only
able to induce lymphangiogenesis but also that this leads
to enhanced lymphatic metastasis [10-12]. Tumour lym-
phangiogenesis can be studied by various parameters like
estimation of expression of VEGF family gene products
by immunohistochemistry or quantitative RT-PCR and
by lymphatic vessel density assessment using antibodies
against proteins specifically expressed on lymphatic en-
dothelium. Several lymphatic endothelial markers have
been established recently like podoplanin, desmoplakin,
Prox 1, receptors for VEGF-C and VEGF-D (VEGFR-3).
Podoplanin is a 38-KD surface glycoprotein expressed on
lymphatic but not blood vascular endothelium has been
demonstrated in the skin. Non specific staining of blood
vessels has been reported [13]. Desmoplakin is a glycol-
protein located exclusively to the intracellular junctions
between the endothelial cells of lymphatic vessels [14].
Antibodies against desmoplakin have indicated specific-
ity for lymphatic endothelium in human tongue but fur-
ther studies are required to confirm the distinctive nature
of desmoplakin staining in other tissue types. Prox-I is
required for the regulation of lymphatic vascular devel-
opment from pre-existing embryonic veins [15]. Anti-
bodies against human Prox-1 to visualize lymphatic ves-
sels in tumour sections have only been used in a limited
number of studies. The vascular endothelial growth fac-
tor receptor 3 (VEGFR-3) is a tyrosine kinase that is
predominantly expressed in Lymphatic endothelial cells
in adult tissues, this marker is not reliable for discrimi-
nating between lymphatic and blood vascular endo the-
lium. Several studies have shown a worse prognosis for
tumours with high lymphangiogenic activity [16,17].
This was a preliminary study of lymphangiogenesis in
35 carcinoma breast patients using D2-40 endothelial
marker for estimation of LMVD and determining its
prognostic significance. A significant correlation be-
tween D2-40 detected LMVD with other unfavorable
prognosis parameters was found. This was in consistence
with studies done in malignant melanoma and bladder
cancer where a significant correlation between LMVD
with the occurrence of lymphatic metastases and survival
was observed [16,17]. Tumour cells are carried to re-
gional lymph nodes through the lymphatics as tumour
emboli. Thus, the detection of peritumoural lymphatic
vessels and its invasion (LVI) on histological sections
can predict lymph node metastasis of the disease in
clinically node negative patients and can serve as an im-
portant prognostic marker.
Histological sections are routinely studied on H & E
staining but it has a limitation of poor yield in lymphatic
vessel visualization and detection of invasion. This non
visualization of lymphatics is because of non staining of
the lymphatic endothelial cells. Majority of tumour em-
boli completely obliterate the lumen of the lymphatics
and they can not be differentiated from nests of tumour
cells which are tumour retraction artifacts and isolated
tumour aggregate due to tissue shrinkage during fixation
in routine H & E staining. Contrary to this D2-40 selec-
tively stains the lymphatic endothelium. This method is
suitable for enhanced visualization of lymphatics as well
as for accurate detection of LVI. In a study done by Kahn
and Marks on 50 breast cancer cases, there was an in-
crease of 18% in LVI detection on D2-40 when com-
pared to H & E staining [18]. Marinho et al. showed

Lymphangiogenesis as a Prognostic Marker in Breast Cancer Using D2-40 as Lymphatic Endothelial
Marker—A Preliminary Study
819
similar detection rates with 28.5% and 13.8% of LVI on
D2-40 and H & E staining respectively [19]. Our result
with D2-40 detected LVI was 37.1% compared to 17.1%
by routine H & E staining. Thus there was a two fold
increase of LVI detection rate using D2-40. The kappa
score obtained in the study showed poor agreement be-
tween LVI detection on D2-40 immunostaining com-
pared with H & E stained sections (k = 0.244).
4.1. Prognostic Parameter Correlation
4.1.1. Lymph Nodes
It was emphasized that a correlation between LMVD and
LVI coexists [20]. The study observed that mean LMVD
in LVI positive and negative cases to be 12 ± 4.2 and 8.3
± 4.2 (p = 0.001). This significant association between
LMVD and LVI could be explained through a lymphan-
giogenesis-induced increase of the “lymphatic window”
providing tumour cells with more opportunities to enter
into lymphatic vessels. The present study observed a
similar result with mean LMVD in LVI positive cases of
22.85 ± 13.29 and in LVI negative cases of 7.95 ± 2.05
(p ≤ 0.001). This signifies that a higher mean LMVD to
be present in LVI positive cases as compared to LVI
negative cases. The result suggests that breast cancers
with high peritumoural lymphangiogenesis as measured
with LMVD more often invade these lymphatic vessels
and have more chance of lymphatic metastasis as com-
pared to patients with low LMVD. This is further sup-
ported with the observation that 85.7% patients with me-
tastatic deposits in the axillary lymph nodes had LMVD
score more than the median (p = 0.030). Further, a sig-
nificant correlation between the total number of positive
lymph nodes and LMVD (r = 0.863, p ≤ 0.0001) also
supports the statement. Similar results were obtained by
Xie et al. on 102 patients of breast cancer where they
found correlation of LMVD with the number of metas-
tatic lymph node (r = 0.964, p < 0.01) [21].
Tumor associated lymphatic vessels in breast cancer are
considered as the main route of tumour cells to axillary
lymph nodes and tumour cells exposed to more microves-
sels are more likely to spread to distant sites and to lymph
nodes [22]. A significant correlation between the LVI posi-
tivity and metastatic lymph node status was observed in the
study (p = 0.011). LVI positivity increased from 7.1% in
patients with no nodal metastasis to 88.8% in patients >10
nodes metastasis. The study suggests that LVI alone can
predict metastatsis to axillary lymph node. Similar results
were observed by Kahn et al. who demonstrated LVI posi-
tivity in 44% of node negative and in 86% of node positive
breast cancer patients [18]. Gurleyik et al. in study of 81
patients showed that when axillary involvement progressed
from negative to more than ten nodes, the rate of positive
LVI increased from 14% to 100% [23].
4.1.2. Tumour Size, Grade and Receptor Status
Tumour size is another important prognostic marker in
breast cancer and predictor of local recurrence, regional
and/or systemic spread and overall survival [24,25]. A
positive correlation between tumor size and LMVD was
suggested [4,26]. Similar observations were found in the
present study where the mean tumour size in patients
with high LMVD was significantly more than those with
low LMVD.
It is speculated that fast growing tumours produce
more growth factors and offer a bigger clonal variety of
tumour cells capable of involving lymphatic vessels
compared with well differentiated slow growing tumours.
Thus it is expected to have a high LMVD associated with
low histologic differentiation grade. Schoppmann et al.
and Kato et al. have shown a significant inverse correla-
tion between grade of tumour with LMVD [15,20]. In the
present study out of 20 patients with high LMVD, 85%
were Grade III and 15% grade II and while out of 15 low
LMVD patients 46.7% were grade III, 46.7% grade II
and 6.7% grade I (p = 0.046). An increasing trend of LVI
positivity with increasing grade of tumour is suggested.
None of the grade I tumour patients was LVI positive,
compared to 50% LVI positivity in grade III tumour pa-
tients (p = 0.066). Conversely, 92.3% of LVI positive
cases were of histological grade III. Gurleyik et al. re-
ported that LVI positivity increased up to 73% when the
tumour grade was III, whereas there were no positive
LVI in grade I tumours [23].
It is true that aggressive tumours have negative recep-
tor status and this should reflect in terms of LMVD and
LVI also, but studies have reported different results of
receptor status with respect to LVI and LMVD and no
unanimity exists in this regard, Gurleyik et al. reported
positive LVI in 85% of estrogen receptor negative tu-
mours (p < 0.0001) and in 73% of progesterone receptor
negative tumours (p = 0.0004) [23]. However, Kato et al.
found no significant correlation between ER/PR status
and LMVD [15]. Schopmann et al. also reported positive
LVI in only 28.2% patients out of 78 estrogen receptor
negative patients (p = 0.961) [20]. Similar to these find-
ings the present study found high LMVD in 36.8% (p =
0.907) and positive LVI in only 52.6% (p = 0.534) of
estrogen and progesterone receptor negative tumours.
4.1.3. Metastatic Poten ti al
The risk of developing lymph node metastasis increases
significantly with the presence of lymphovascular inva-
sion and hence it can be regarded as the predictor of
nodal involvement and also the disease free survival.
Schoppmann et al. [20] and Kato et al. [15] compared
disease free survival with LMVD and failed to find a
significant correlation. Schopmann et al. [20] found a
significant difference in overall survival and disease free

Lymphangiogenesis as a Prognostic Marker in Breast Cancer Using D2-40 as Lymphatic Endothelial
Marker—A Preliminary Study
820
survival between patients with or without LVI both in
univariate and multivariate survival analysis. In the pre-
sent study five out of thirty five patients developed me-
tastasis in follow up. Though a significant correlation
between disease free survival and LMVD could not be
drawn yet the mean LMVD in patients who developed
metastasis was higher as compared to those who were
free of metastasis (21.6 ± 12.66 and 12.13 ± 10.16 re-
spectively) (p = 0.071). On the other hand the LVI posi-
tivity was significantly higher in patients who developed
metastasis (p = 0.018). The observation favors the state-
ment that the patients with lymphatic embolization are
more prone for metastatic disease.
5. Conclusion
The system of lymphangiogenesis represents a potential
new target for cancer prognostication and development
of anti-cancer strategies. Specific lymphatic endothelial
markers, such as podoplanin, Prox-1, and LYVE-1, now
provide sufficient tools to researchers to understand bet-
ter, the concepts of tumour lymphangiogenesis. The data
presented herein support the importance LVI and LMVD
assessment using D2-40 in breast cancer patients for
prognostic purpose. The higher positivity of LMVD and
LVI correlated with other established prognostic meas-
ures like tumour size, lymph node metastasis, number of
involved nodes, grade of tumour and tumour metastasis.
This highlights the use of this novel marker in identifica-
tion of patients who will have a poor prognosis even if
they have early cancer without nodal involvement. LMVD
and LVI assessment using D2-40 can be used as a single
prognostic marker in primary breast cancer but needs
multicentric study and a longer follow up for its validity
which can probably establish the entity.
6. Acknowledgements
The authors acknowledge the contribution of Prof Saroj
Gupta for her contribution in pathological examination.
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