Lymphangiogenesis as a Prognostic Marker in Breast Cancer Using D2-40 as Lymphatic Endothelial Marker—A Preliminary Study

doi:10.4236/jct.2012.325103

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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.

Email: [email protected], sunn[email protected], vivekims97@gmail.com, [email protected],

[email protected], #[email protected]

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

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

III

1 (2.9)

10 (28.6)

24 (68.6)

No. of involved Lymph Nodes

1 - 3

4 - 9

>10

14 (40.0)

7 (20.0)

5 (14.3)

9 (25.7)

Positive

Negative

3 (13.63)

19 (86.36)

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)

0.388

Positive lymph node

1 - 3

4 - 9

>10

3 (100)

4 (22.2)

5 (27.8)

9 (50.0)

0.030

Grade

III

1 (6.7)

7 (46.7)

3 (15.0)

17 (85.0)

0.046

Positive

Negative

1 (12.5)

7 (87.5)

2 (14.3)

12 (85.7)

0.907

Positive

Negative

1 (12.5)

7 (87.5)

2 (14.3)

12 (85.7)

0.907

HER

Positive

Negative

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)

0.755

Grade

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

Positive

Negative

1 (9.1)

10 (90.9)

2 (18.2)

9 (81.8)

0.534

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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