Evaluation of Lympho-vascular Invasion in Breast Carcinoma Using Lymphatic Endothelial Marker (D2-40)

Breast cancer has become the most frequently diagnosed cancer and the leading cause of cancerrelated death among women worldwide, overtaking lung cancer. The Present study attempts to investigate the use of D2 -40 for the detection of lymphovascular invasion in node negative breast cancer. Additionally the lympho-vascular invasion using D2 -40 and its association with distant metastasis and overall prognosis of the patient was also explored.


INTRODUCTION
The malignant tumour that has developed in the cells of the breast is called breast carcinoma or breast cancer. Breast carcinoma has been extensively studied and with the availability of vast evidence based data [1] and literature, various treatment modalities have been introduced to cure this disease. Breast cancer has become the most frequently diagnosed cancer and the leading cause of cancer-related death among women worldwide, overtaking lung cancer. Although incidence rates are higher in the West, the highest burden for breast cancer is

Original Research Article
in the developing countries [2]. Majority of the breast carcinomas are usually asymptomatic and the usual mode of presentation is an incidental palpable lump or pain and rarely, they present with nipple discharge and skin changes [3]. Breast carcinomas have varying levels of invasion and aggressiveness irrespective of the duration of presentation. Mammary glands, also known as breasts are modified sweat glands [4][5][6]. In utero, breast development starts in the first trimester of gestation with multiple bilateral thickenings of the ectoderm on the ventral aspect of the fetus [5][6][7][8][9][10]. This thickened ridge extends in a linear fashion from the axilla to the groin, forming the milk line [11]. The breasts begin to develop at puberty. This development is stimulated by the estrogen-both mammary glands plus the deposition of fat. During pregnancy, large quantities of estrogen is secreted by the placenta which cause the ductal system of the breasts to grow and branch [12].
For the breasts to develop into milk -secreting organs also requires progesterone. Once the ductal system has developed, progesterone starts acting synergistically with estrogen causing additional growth of the breast lobules along with budding of alveoli and development of secretory characteristics in the cells of the alveoli [13].They are a group of malignant epithelial tumours which are characterized by invasion of adjacent tissues and have an increased tendency to metastasize to various sites [14]. The majority of them are adenocarcinomas. They exhibit a wide range of morphological and specific histopathological subtypes and most of them have specific prognostic or clinical characteristics [15]. Breast cancer in women is a major health burden globally. There are about 2.1 million newly diagnosed female breast cancer cases in 2018, accounting for almost 1 in 4 cancer cases among women. The incidence rates are highest in Australia/New Zealand, Northern Europe, Western Europe with Belgium being the highest in global rates, and the Netherlands, and France, Southern Europe (Italy), and Northern America [16]. Myoepithelial cells appear to arise from basal cells between 23-28 weeks of gestation and they play an important role in the morphogenesis of the mammary gland through the synthesis of basement membrane constituents such as laminin, type IV collagen, and fibronectin, as well as metalloproteinases and growth factors. In the last 2 months of gestation, the epithelial columns branch, canalize, and transform into ducts (and eventually into lobules). A 'pit' in the epidermis forms at the convergence of the major lactiferous ducts, and thereafter, its eversion forms the protuberant nipple [11].
Even though extensive screening programs and clinical tests are available for early detection of this disease, there is higher morbidity and mortality rate of breast cancer in developing countries due to increased socioeconomic dependence and delayed diagnosis [4]. There is a lack of self-awareness of this life threatening malignancy in our country.
Triple assessment including clinical examination, imaging (mammography and ultrasound) and tissue sampling by either fine needle aspiration cytology or needle core biopsy helps in evaluating breast abnormalities [17]. A systematic clinical examination should be done taking into account the nature of the lump and, if present, any skin dimpling or change in contour of the breast and also assessment of the axilla is very important 4.
Lymph node metastasis is considered as one of the most important prognostic factors for breast carcinomas [3]. So, the recognition of lymphatic vessel invasion in histopathological sections is crucial. Histopathological correlation along with analysis by immunohistochemistry (IHC) has instigated various modalities of treatment. The standard norm of management for breast cancer includes surgery, chemotherapy and radiotherapy depending on the various parameters [5]. The breasts are supplied by branches of the axillary artery, the internal thoracic artery and some intercostal arteries. The second perforating artery, a branch of anterior intercostal artery is usually the largest, and supplies the upper region of the breast, nipple, areola and adjacent breast tissue [11,12].
Interest in D2-40 first instigated its use as a lymphatic endothelial marker, since it does not stain vascular endothelium. As such, D2 -40 immuno-stains have been used as lymphatic endothelial marker to study lymph-angiogenesis in breast carcinoma [18] and has been proved to be helpful in determining lymphatic invasion by the tumour [19].

Source of Data
The present study was undertaken in the Department of Pathology, Sree Balaji Medical College & Hospital, Chennai.

Type of Study
Cross sectional study.

Inclusion criteria
Only histopathologically confirmed cases of invasive carcinoma of breast will be included in the study

Exclusion criteria
• Benign and inflammatory lesions of the breast will be excluded.
• Patient who has received adjuvant chemotherapy will be excluded from the study

Descriptive statistics 2. Contingency table analysis using SPSS for
Windows (version 24.0) 3. P <0.005 is considered as statistically significant.

Method of Collection of Data
Purposive sampling technique (26): All mastectomy specimens of invasive breast carcinoma were studied noting the clinical details. The formalin fixed tissue were subjected to routine processing and sections stained with Haematoxylin & Eosin (H&E) studied extensively for all the prognostic factors.
The grading of breast carcinoma was done according to the Nottingham combined histologic grade (Elston Ellis modification of Scarff Bloom Richardson grading system) [20].
And patients were divided into three NPI groups [22]. Immunohistochemistry: Sections from each block was subjected to IHC staining with D2-40 (Monoclonal mouse antihuman antibody, clone PM231, PathnSitu).   Slides were then washed in tap water for 3 -5minutes followed by distilled water.

Procedure
 Endogenous peroxidase was quenched by dipping slides into a fresh aqueous solution of 3% peroxide for 15 minutes  Slides were then washed in tap water for 3 -5 minutes followed by distilled water.
 Then slides were rinsed with Tris buffer for each 5 minutes, three changes.

Detection of Antigens in Paraffin sections
 The primary antibody was added at a dilution selected usually for half an hour to 1hour.

Lymphatic Vessel Density Assessment
Determination of LVD was performed according to Weidner et al. [23]. The immuno-stained sections were scanned by light-microscopy at low magnification (x40 & x100) and the areas of tissue with the greatest number of distinctly highlighted lymph vessels ('hot spots') were selected. LVD was then assessed by counting all immuno-stained vessels at a total magnification of x400 from three areas for each case.
The mean number of lymph vessels in each case was counted. Both the scoring and counts were performed blindly without any prior clinical knowledge of the patients. LVI was considered evident if at least one tumour cell cluster could be clearly visualised inside the D2-40 positive lymphatic space.The LMD ranged from 0 -16 micro-vessels/hpf and the mean was calculated as 5.4. Anything above it was considered as increased microvessel density.

Age at the Onset of Disease
Of 30 cases, the maximum number of cases 16/30 (53.3%) was in the age group of in the age group 30 to 40 years, followed by 6 cases (20%) in the age group 61 -70 years and 4 cases (13.3%) each in the age group 41 to 50 and 51 to 60 years. The youngest patient in our study was 36 years old and the eldest was 70 years old.

Site of Involvement
Of 30 cases, majority of them were located in upper outer quadrant (30%), followed by upper inner quadrant (26.7%), lower inner quadrant (20%), and lower outer quadrant (16.7%), one central and diffuse (3.3%) in the order of decreasing frequency.

Side of Involvement
Out of 30 cases, 50% had tumour on left side and 50% had tumour on right side.

Histological Grade of the Tumour
30 cases of mastectomy specimens when graded based on Nottingham modification of the Scarff-Bloom-Richardson grading system, and it showed a predominance of grade 3 tumours (66.6%) compared to the grade 2 tumours (26.7%) followed by grade 1 tumours (6.7%).

Associated Morphological Parameters
Lymphocytic infiltration (Fig. 2) was observed in 62.9% of cases while necrosis (Fig. 3) was seen in 58.1% of cases respectively.

TNM Staging of the Tumour
Staging of the tumours was done using AJCC system of classification based on tumour (T), node (N) and Metastasis (M).
Majority of the cases 21/30 (70%) were of pT2 with tumours being more than 2cm but less than 5cm in their greatest dimension. There were 8/30 (26.7%) of pT3 (tumour more than 5 cm in greatest dimension) and 1/30 (3.3%) of pT1 (tumours being < 2cm in their greatest dimension). There were no cases of pT4 (tumour of any size with direct extension to skin/chest wall).
100% cases having tumour size of 5cm were poorly differentiated tumours (Grade 3), 66.7% cases having tumour size of 2-5 cm were Grade 3 and 25% cases with tumour size 2 -5cm were of Grade 2. There was positive correlation between increased tumour size and higher histological grade, though not statistically significant. There was a significant association between tumours with increased number lymph node metastasis and higher histological grade.

Nottingham's Prognostic Index Groups
Patients were divided into three NPI groups based on Nottingham's Prognostic index (NPI) calculated by the formula,

D2-40 stained LVI associated with tumour size
All tumours of size >5 cm showed D2 -40 stained LVI (100%) and 14 cases (58.3%) of tumours of size 2 -5 cm also showed LVI, while 10 cases showed no LVI. There was significant association between D2 -40 expression and greater size of tumour.
Out of 30 cases, lymph node metastasis was seen in 23 cases (76.7%) and LVI was seen in 20 cases (66.7%). Tumours with increased number of lymph node metastasis showed increased LVI.

Age Distribution
In the present study, the age range of the patients with invasive breast carcinoma varied from 36 years to 70 years with a mean age of 48.1 years. LVI was seen in 20 cases of which 7 cases (35%) were > 40 years and 13 cases (65%) were < 40 years.
Various studies have demonstrated that younger age is a risk factor for LVI and axillary lymph node metastasis [31,32]. This has been attributed to biologically more aggressive tumours in this younger age group [33].

Correlation of Tumour Size with D2 -40 Expression Andcomparison with Other Studies
Various studies have shown that the size of tumour is one of the most significant prognostic factors in breast carcinoma and there is increased incidence of axillary lymph node metastasis and decreased survival with increasing size of the tumour.
In our study, tumour size varied from 2 cm to 8 cm with a mean of 3.88 cm. Maximum number of tumours were in the range of 2cm to 5 cm (T2) (66.7%) followed by tumours of size >5 cm (T3) (26.6%) and of <2 cm (T1) (6.7%).
Studies have confirmed that LVI is significantly lower in tumours ≤2 cm than that in tumours>2 cm. These studies were in concordance with our findings.

Correlation of Histological Tumour Grade with LVI and Comparison with Other Studies
Histologic grading has become widely accepted as a powerful indicator of prognosis in breast carcinoma and its importance has been validated by multiple independent studies. High grade and fast growing tumour may produce more growth factors and offer a bigger clonal variety of tumour cells capable of invading lymphatic vessels compared with low grade and slow growing tumour [38].
In the present study, there is a significant association of LVI in grade 3 tumours (81.8%) when compared to grade 2 tumours (33.3%). This was in concordance with these following studies.

Correlation of Lymph Node Status with Lympho-vascular Invasion
In our study, D2-40 expression correlated significantly with node positive tumours. Axillary lymph nodes were positive for metastasis in 66.7% of cases. In cases with higher lymph node metastasis, i.e. N2 and N3 had increased LVI and LMVD stained by D2 -40 in 83.3% and 100% of cases, respectively.
Rakha EA et al. [26] reported that presence of LVI in LN negative tumours should be considered prognostically equivalent to those cases with 1-3 positive LNs (pN1) and should recommended to be candidates for neoadjuvant therapy.
Studies mentioned below observed a similar trend with a significant association between LVI stained by D2 -40 and axillary lymph node involvement.

Correlation of Lymphatic Microvessel
Density with

Lympho-vascular Invasion
An increased lymphangiogenesis is found to be associated with increased LN positivity and hence, increased lympho-vascular invasion. Increase in lymph mean vessel density significantly increases the potential  also stated that increased LVD associated with the poor prognostic indicators like larger tumour size, higher grade, lymph node metastasis.

CONCLUSION
A total of 30 cases of invasive breast carcinomas were included in the study. Lympho-vascular invasion was analysed by D2-40 immuno-stain and was correlated with other prognostic factors. A significant trend was noted with D2 -40 stained lymphovascular invasion and poor prognostic factors including larger tumour size, lymph node metastasis, and higher histological grade. Vascular drainage upsurges the chance of visceral metastasis resulting in the circulating tumour cells having a greater potential of distal implantation. The association between lymphovascular invasion and the distant metastasis signify that early detection of LVI can be used as an independent poor prognostic marker in breast carcinoma patients with lymph node-negativity. Also, this study reinforces the significance of pathologic analysis in the prognostic evaluation of breast cancer, especially those factors that cannot be analysed on molecular level, such as size, nodal status, and LVI positivity. Presence of LVI was consistently associated with reduced disease-free interval (DFS) and overall survival (OS), regardless of tumour and treatment characteristics in the high-risk group.In addition, moderate to marked LVI has to be an indicator of postoperative irradiation after conservative breast surgery in node negative patients.
D2-40 has been proven to be a valuable marker in identifying lympho-vascular invasion in various studies. So, we propose the consistent use of D2 -40 immunostain while evaluating the lymphatic invasion by tumour cells for further follow-up and overall prognosis. The effect of neoadjuvant therapy on LVI is out of the concept of this study, and in order to have a uniform patient group, we excluded the patients who had undergone neoadjuvant therapy.