|Year : 2018 | Volume
| Issue : 2 | Page : 176-180
|Comparison of lymphangiogenesis, lymphatic invasion, and axillary lymph node metastasis in breast carcinoma
Prerna Guleria1, V Srinivas2, D Basannar3, Vibha Dutta4
1 Department of Pathology, Military Hospital Yol Cantt, Kangra, Himachal Pradesh, India
2 Department of Pathology, Command Hospital (Southern Command), Pune, Maharashtra, India
3 Department of PSM, AFMC, Pune, Maharashtra, India
4 Department of Pathology, Command Hospital (Central Command), Lucknow, Uttar Pradesh, India
Click here for correspondence address and email
|Date of Web Publication||20-Apr-2018|
| Abstract|| |
Context: Lymphangiogenesis correlates with poor prognosis in Invasive Ductal Carcinoma (IDC) breast. D2-40 antibody, a specific marker for lymphatic endothelium, differentiates lymphatic from vascular endothelium. Therefore, the aims of this study were to estimate lymphangiogenesis using D2-40 antibody and correlate with lymphatic invasion (LI) and axillary lymph node (LN) status and compare lymphatic mean vessel density (LMVD) with Tumor (T) and Node (N) stages and grade of tumor. Methods and Material: The study was conducted on fifty consecutive cases of IDC breast who underwent modified radical mastectomy (MRM) from Jan 2009 to March 2011. Hematoxylin-eosin sections and Immunohistochemistry (IHC) slides were studied along with their LN status. LMVD was counted after D2-40 immunostaining (100x magnification) in three hot spots in peritumoral areas and averaged. LI as opposed to vascular invasion (BVI), and LN status for all cases were assessed. Statistical Analysis: Statistical analysis was done using SPSS software (version 14.0 for Windows). Pearson's correlations, χ2 tests and Mann-Whitney U test were used. Results: Lymphangiogenesis varied from 0 to 58 with mean LMVD of 11. Of 50 cases, five showed no lymphatic vessels in peritumoral areas; of these five, three had positive LNs. 21/50 cases had LI. No statistical significant association was seen between lymphangiogenesis and LI. 34/50 cases had positive LNs. Mean LMVD was higher in patients with N2/N3 stage as compared to N0/N1 stage and was statistically significant (P = 0.013). Conclusions: D2-40 is specific marker for lymphatic endothelium. LI and lymphangiogenesis, as opposed to BVI, are better prognostic indicators in IDC breast.
Keywords: D2-40, Invasive ductal carcinoma breast, lymphangiogenesis
|How to cite this article:|
Guleria P, Srinivas V, Basannar D, Dutta V. Comparison of lymphangiogenesis, lymphatic invasion, and axillary lymph node metastasis in breast carcinoma. Indian J Pathol Microbiol 2018;61:176-80
|How to cite this URL:|
Guleria P, Srinivas V, Basannar D, Dutta V. Comparison of lymphangiogenesis, lymphatic invasion, and axillary lymph node metastasis in breast carcinoma. Indian J Pathol Microbiol [serial online] 2018 [cited 2018 Sep 22];61:176-80. Available from: http://www.ijpmonline.org/text.asp?2018/61/2/176/230566
| Introduction|| |
Metastasis is the leading cause of mortality in patients of breast cancer (BC) and other solid tumors. Frequently, the initial sites of metastasis are the regional lymph nodes (LNs)., Migration of tumor cells into the LNs is greatly facilitated by lymphangiogenesis, a process that generates new lymphatic vessels from preexisting lymphatics , or from lymphatic endothelial progenitors. This process is dynamic during embryogenesis but is relatively rare in adulthood. Lymphangiogenesis after staining with specific markers is found in greater numbers in regions with BCs as compared to normal breast parenchyma., An increased lymphangiogenesis is found to be associated with increased LN positivity.,
Lymphatic metastasis may not exclusively depend on the generation of new vessels, although undoubtedly, increase in lymph mean vessel density (LMVD) significantly increases the potential for a tumor cell to invade lymphatic vessel surface. This may account for strong statistical associations between lymphovascular invasion (LVI) encompassing both preexisting and new vessels and LN metastasis., It is possible, however, that in some cases, breast tumors fail to induce lymphangiogenesis so that lymphatic metastasis occurs only through the preexisting vessels. New lymphatic vessels may provide an additional track for tumor cell transit, thus bolstering the prometastatic activity of the preexisting lymphatic vessels situated at the tumor border. This fact led to the concept of quantification of tumor lymphangiogenesis (LMVD). LMVD quantification is much more challenging than blood vessel density because of the natural heterogeneous distribution of lymphatic vessels. In contrast to blood vessels, lymphatic vessels also support spread of metastatic cells but not tumor cell proliferation and expansion of the tumor mass. Therefore, subtle increases in LMVD might be missed in tumor sections set aside for immunohistochemical analysis although they might suffice for tumor dissemination in a patient. A consensus seems to exist that increased density of peritumoral lymphatic vessels might be sufficient for tumor cell transit to LN even in the absence of intratumoral lymphatics.
Another aspect which has gained attention is the presence of LVI in the tumor periphery. LVI encompassing both types of vessels, i.e., lymphatic and blood vessels has long been recognized as a poor prognostic indicator. However, early studies did not distinguish between the two due to the absence of specific lymphatic markers. The emergence of specific lymphatic markers, such as LYVE-1, podoplanin/D2-40, and Prox, has enabled clear distinction between lymphatic vessel invasion (LI) and blood vessel invasion (BVI). The preferential spread of tumor cells through lymphatic vessels might stem from the high frequency of LI in BC as compared with BVI. In patients with clinically staged I and II breast tumors, LN status is one of the most important prognostic factors for survival independent of tumor size, histological grade, and other clinicopathological parameters.,
Strong associations have been found among tumor-induced lymphangiogenesis, LVI, regional lymphatic spread, distant metastasis, and survival. Therefore, inhibition of tumor lymphatic vessels appears to be an attractive target to prevent metastasis to LNs.
Our objectives of the study, therefore, were (a) to estimate lymphangiogenesis using D2-40 monoclonal antibody as a specific marker of lymphatic endothelium, (b) to correlate lymphangiogenesis with LI and axillary LN status, and (c) to compare LMVD with T and N stages and grade of tumor.
| Materials and Methods|| |
- Study population: Fifty consecutive cases of IDC breast who underwent modified radical mastectomy (MRM) at our institute from January 2009 to March 2011 were studied. Archival data and paraffin blocks were retrieved
- Inclusion criteria: The cases were included both genders, any age, and any tumor, node, and metastasis status
- Exclusion criteria: Noncarcinomatous malignancies of the breast, for example, sarcoma, lymphoma, melanoma, recurrent carcinoma(s) breast, metastatic tumors to the breast, and patients of carcinoma breast where axillary LN dissection was not carried out as a part of surgical procedure were excluded from the study
MRM specimens were dissected as per standard protocol. Tissues were formalin fixed and paraffin embedded. H- and E-stained slides from all the blocks of the tumor were reviewed, and the section showing tumor with adjacent normal breast parenchyma was selected. Immunohistochemistry (IHC) using D2-40 to stain lymphatic endothelium was carried out on all cases using standard protocol 
- Assessment of LMVD: Immunostained tumor sections were scanned at low magnification (×10 ocular and × 10 objective), and the three most vascularized areas in the peritumoral areas (hot spots) were chosen. Peritumoral lymph vessels were defined as D2-40-positive vessels located in preexisting mammary stroma within a maximal distance of 2 mm from the tumor periphery. The LMVD was calculated by counting the number of lymphatic channels stained by D2-40 antibody in three hot spots. An average of the three counts was taken as the LMVD for the case.
This study has the approval of the ethics committee of the institution.
Statistical analysis was done using SPSS software version 14.0 for Windows (IBM Corporation, New York, USA. (a) Pearson's correlation, (b) Chi-square test, and (c) Mann–Whitney U-test were used. For analyzing associations between categorical variables (e.g., presence of LI and presence of LN metastases), Chi-square test was used. Where the data were not normally distributed, nonparametric test, i.e., Mann–Whitney U-test was used to compare independent variables. P<0.05 was considered statistically significant.
| Results|| |
All the 50 consecutive cases of IDC breast were female patients. The youngest patient was 30 years of age, and the oldest patient was 72 years of age (mean age 52.7 years). The peak incidence of IDC was seen in the 51–60 year age group. All the tumors were histomorphologically IDC (not otherwise specified [NOS]) [Figure 1]a.
|Figure 1: (a) Infiltrating Ductal Carcinoma, NOS Breast (×100). (b) Peritumoral area showing lymphovascular invasion (×40). (c) D2-40 staining lymphatic vessel with lymphovascular invasion (×100). (d) Tumor emboli completely filling the lymphatic vessel (×400). (e) Tumor island mimicking stromal invasion on H and E and D2-40 immunostain (×400)|
Click here to view
All cases showed the presence of lymphatic channels both in the intratumoral and peritumoral areas. All of these lymphatic channels stained with D2-40 monoclonal antibody, a specific stain of lymphatic endothelium. As no false positives were seen, immunostaining for D2-40 showed a high specificity and positive predictive values. LMVD varied from 0 to 58 with mean LMVD of 11. LMVD of 0 was seen in 5 cases (3 of these 5 cases had positive LNs).
The cases were regrouped further into two groups each according to their T and N stages and grade of the tumor for ease of statistical evaluation. Patients with T stages 1 and 2 were grouped together and those with stages T3 and T4 were combined as the second group. Similarly, N0 and N1 were combined into Group 1 and N2 and N3 as Group 2. Tumors of Grade 1 and 2 were put together into one group, and Grade 3 tumors were kept separate in another group. The mean for LMVD was higher in cases with higher T stages (T3 and T4). However, this was not significant statistically. The median for higher T stages was 10.67 with Mann–Whitney U-test statistics 243 and a P = 0.846.
The mean LMVD was 17.2 in cases with higher N stages (N2 and N3) as compared to 8.76 for lower N stages (N0 and N1). The median for higher N stages was 14.56, with Mann–Whitney U-test statistics 152.5 and a significant P = 0.013. Therefore, higher N stages had statistically significant higher LMVD. The mean LMVD was higher in cases with higher grade of tumor (Grade 3) but was not statistically significant. The median for higher grade tumors was 11.67, with Mann–Whitney U-test statistics 211 and a P = 0.375.
LVIs were assessed in the peritumoral area only [Figure 1]b. About 55.3% of cases with LVI on H and E stained positively for D2-40 antibody [Figure 1]c, thereby indicating that the tumor embolus was actually within a lymph vessel, i. e., LI. The mean LMVD in cases with LI was higher than in cases with no LIs. However, this was not statistically significant. The median for cases with LIs was 9.33, with Mann–Whitney U-test statistics 297 and P = 0.883. Patients with positive LNs had a higher mean LMVD, but this was not statistically significant. The median LMVD in positive LNs was 10.33, with Mann–Whitney U-test statistics 231.5 and P = 0.399. The correlation of all clinicopathological parameters with D2-40 immunostaining is shown in [Table 1].
D2-40 also stained the myoepithelial cells (MEC); however, the staining pattern was different. The MECs stained less intense with an irregular staining pattern as compared to lymphatic endothelium. The antibody also stained few tumor emboli which completely filled the lymph vessel as shown in [Figure 1]d. One case where a perineural invasion on H and E was misdiagnosed as stromal invasion stood out as LI after D2-40 immunostaining [Figure 1]e.
| Discussion|| |
IDC is the most common morphological variant seen amid BCs. Our study included 50 consecutive cases of BCs, all of which were IDC (NOS) breast. BC is one of the leading causes of death in women between the ages of 35–60 years. The mean age of 52.7 years was seen in our study. BCs have a female predominance and are rare in males as seen in the epidemiological study carried out by Yeole and Kurkure, where it was seen that BC is about hundred times more common among women than among men. All 50 cases included in this study were females.
D2-40 monoclonal antibody is a specific and preferred immunostain for lymphatic endothelium., The intratumoral and peritumoral lymphatic vessels in all our cases stained positively for D2-40 antibody. Therefore, D2-40 had 100% specificity in our study. This compares favorably with the study by Kahn and Marks  who also found D2-40 to be highly specific and sensitive. LVIs were studied at the peritumoral areas denoting the malignant front of tumor and also because the lymphatics are more in the peritumoral area, as seen in other studies.,,,
Lymphangiogenesis is present in BC patients as was seen in our study. Several other studies have also brought out this fact., However, Boneberg et al. in their study contradicted this by saying that primary BCs are not a site of highly active angiogenesis and lymphangiogenesis. There have been studies showing association of lymphangiogenesis with LN metastases.,, Lymphangiogenesis is also seen to be increased in cases of triple-negative BC. It has also been associated with extensive lymphatic spread of inflammatory BC. It has also been seen that inhibiting human epidermal growth factor receptor 2/neu reduces lymphogenic metastasis.
In our study, lymphangiogenesis was significantly associated with higher N stages. This is in concordance with the study conducted by Kandemir et al. as well as Ansari et al., Kandemir et al. had also found a significant correlation between lymphangiogenesis and higher T stages, LI and LN status, and Ansari et al. had, in addition, found significance while correlating it with the grade of tumor. Our study also showed a similar trend though we did not find any statistical significance. This may be attributed to the small sample size of only 50 cases. Further studies comprising larger study population are warranted to statistically confirm the fact. An important aspect of detecting lymphangiogenesis is the concept of targeted therapy. Studies have shown that targeted therapy may be beneficial in preventing metastasis and combat advanced cancer.
| Conclusion|| |
Our study shows D2-40 monoclonal antibody to be a specific immunostain for lymphatic endothelium and helps detect LIs. Lymphangiogenesis is seen in IDC breast, and although LMVD increased as T stage and grade increased, it was not statistically significant. Detection of lymphangiogenesis may be a potential for targeted therapy as our study shows that LMVD increased significantly with higher N stages.
We would like to thank Professor and HOD, Department of Pathology, AFMC, Pune.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Schoppmann SF, Horvat R, Birner P. Lymphatic vessels and lymphangiogenesis in female cancer: Mechanisms, clinical impact and possible implications for anti-lymphangiogenic therapies (Review). Oncol Rep 2002;9:455-60.
Kyzas PA, Geleff S, Batistatou A, Agnantis NJ, Stefanou D. Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma. J Pathol 2005;206:170-7.
Chua B, Ung O, Taylor R, Boyages J. Frequency and predictors of axillary lymph node metastases in invasive breast cancer. ANZ J Surg 2001;71:723-8.
Cunnick GH, Jiang WG, Gomez KF, Mansel RE. Lymphangiogenesis and breast cancer metastasis. Histol Histopathol 2002;17:863-70.
Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, et al.
Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med 2001;7:192-8.
He Y, Karpanen T, Alitalo K. Role of lymphangiogenic factors in tumor metastasis. Biochim Biophys Acta 2004;1654:3-12.
Cunnick GH, Jiang WG, Douglas-Jones T, Watkins G, Gomez KF, Morgan MJ, et al.
Lymphangiogenesis and lymph node metastasis in breast cancer. Mol Cancer 2008;7:23.
Boneberg EM, Legler DF, Hoefer MM, Ohlschlegel C, Steininger H, Füzesi L, et al.
Angiogenesis and lymphangiogenesis are downregulated in primary breast cancer. Br J Cancer 2009;101:605-14.
Choi WW, Lewis MM, Lawson D, Yin-Goen Q, Birdsong GG, Cotsonis GA, et al.
Angiogenic and lymphangiogenic microvessel density in breast carcinoma: Correlation with clinicopathologic parameters and VEGF-family gene expression. Mod Pathol 2005;18:143-52.
El-Gohary YM, Metwally G, Saad RS, Robinson MJ, Mesko T, Poppiti RJ, et al.
Prognostic significance of intratumoral and peritumoral lymphatic density and blood vessel density in invasive breast carcinomas. Am J Clin Pathol 2008;129:578-86.
Williams CS, Leek RD, Robson AM, Banerji S, Prevo R, Harris AL, et al
. Absence of lymphangiogenesis and intratumoural lymph vessels in human metastatic breast cancer 6. J Pathol 2003;200:195-206.
Ran S, Volk L, Hall K, Flister MJ. Lymphangiogenesis and lymphatic metastasis in breast cancer. Pathophysiology 2010;17:229-51.
Colleoni M, Rotmensz N, Maisonneuve P, Sonzogni A, Pruneri G, Casadio C, et al.
Prognostic role of the extent of peritumoral vascular invasion in operable breast cancer. Ann Oncol 2007;18:1632-40.
Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, et al.
LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 1999;144:789-801.
Mohammed RA, Martin SG, Gill MS, Green AR, Paish EC, Ellis IO, et al.
Improved methods of detection of lymphovascular invasion demonstrate that it is the predominant method of vascular invasion in breast cancer and has important clinical consequences. Am J Surg Pathol 2007;31:1825-33.
Kahn HJ, Marks A. A new monoclonal antibody, D2-40, for detection of lymphatic invasion in primary tumors. Lab Invest 2002;82:1255-7.
Wilting J, Papoutsi M, Christ B, Nicolaides KH, von Kaisenberg CS, Borges J, et al.
The transcription factor pro×1 is a marker for lymphatic endothelial cells in normal and diseased human tissues. FASEB J 2002;16:1271-3.
Grabau D, Jensen MB, Rank F, Blichert-Toft M. Axillary lymph node micrometastases in invasive breast cancer: National figures on incidence and overall survival. APMIS 2007;115:828-37.
Sivridis E, Giatromanolaki A, Galazios G, Koukourakis MI. Node-related factors and survival in node-positive breast carcinomas. Breast 2006;15:382-9.
Boenisch T. Dako Handbook of Immunohistochemical Staining Methods. 3rd
ed. California: Dako Corporation; 2001.
Yeole BB, Kurkure AP. An epidemiological assessment of increasing incidence and trends in breast cancer in Mumbai and other sites in India, during the last two decades. Asian Pac J Cancer Prev 2003;4:51-6.
Arnaout-Alkarain A, Kahn HJ, Narod SA, Sun PA, Marks AN. Significance of lymph vessel invasion identified by the endothelial lymphatic marker D2-40 in node negative breast cancer. Mod Pathol 2007;20:183-91.
Ito M, Moriya T, Ishida T, Usami S, Kasajima A, Sasano H, et al.
Significance of pathological evaluation for lymphatic vessel invasion in invasive breast cancer. Breast Cancer 2007;14:381-7.
Kulkarni D, Shah AA, Ingle Y, Ramaiah VV, Goswamy K, Naz-Muddebihal F, et al
. To study the regional difference in intra and peri-tumoral lymphangiogenesis in various histopathological grades and stages of oral squamous cell carcinoma and correlating with lymph node metastasis using D2-40: An immunohistochemical study. J Int Oral Health 2016;8:422-8. [Full text]
Zhang XH, Huang DP, Guo GL, Chen GR, Zhang HX, Wan L, et al.
Coexpression of VEGF-C and COX-2 and its association with lymphangiogenesis in human breast cancer. BMC Cancer 2008;8:4.
Van den Eynden GG, Van der Auwera I, Van Laere SJ, Colpaert CG, van Dam P, Dirix LY, et al.
Distinguishing blood and lymph vessel invasion in breast cancer: A prospective immunohistochemical study. Br J Cancer 2006;94:1643-9.
Liu HT, Ma R, Yang QF, Du G, Zhang CJ. Lymphangiogenic characteristics of triple negativity in node-negative breast cancer. Int J Surg Pathol 2009;17:426-31.
Van der Auwera I, Van den Eynden GG, Colpaert CG, Van Laere SJ, van Dam P, Van Marck EA, et al.
Tumor lymphangiogenesis in inflammatory breast carcinoma: A histomorphometric study. Clin Cancer Res 2005;11:7637-42.
Schoppmann SF, Tamandl D, Roberts L, Jomrich G, Schoppmann A, Zwrtek R, et al.
HER2/neu expression correlates with vascular endothelial growth factor-C and lymphangiogenesis in lymph node-positive breast cancer. Ann Oncol 2010;21:955-60.
Kandemir NO, Barut F, Bektas S, Ozdamar SO. Can lymphatic vascular density be used in determining metastatic spreading potential of tumor in invasive ductal carcinomas? Pathol Oncol Res 2012;18:253-62.
Ansari MA, Srivastav V, Sahoo M, Kumar M, Kumar A. Prognostic evaluation of angiogenesis and lymphangiogenesis using endothelial markers in breast cancer. Int J Gen Med Pharm 2014;3:49-56.
Kodera Y, Katanasaka Y, Kitamura Y, Tsuda H, Nishio K, Tamura T, et al.
Sunitinib inhibits lymphatic endothelial cell functions and lymph node metastasis in a breast cancer model through inhibition of vascular endothelial growth factor receptor 3. Breast Cancer Res 2011;13:R66.
Department of Pathology, Military Hospital Yol Cantt, Dharamshala, Kangra - 176 052, Himachal Pradesh
Source of Support: None, Conflict of Interest: None
| Article Access Statistics|
| Viewed||875 |
| Printed||50 |
| Emailed||0 |
| PDF Downloaded||281 |
| Comments ||[Add] |