Oncology Journal of India

: 2020  |  Volume : 4  |  Issue : 1  |  Page : 23--27

Importance of endothelial markers in detection of lymphovascular invasion in carcinoma breast and its correlation with axillary lymph node metastasis

Khurshida Rahman1, Nageswar Sahu1, Urmila Senapati1, Subrat Kumar Sahu2,  
1 Department of Pathology, Kalinga Institute of Medical Sciences, KIIT University, Bhubaneswar, Odisha, India
2 Department of Surgery, Kalinga Institute of Medical Sciences, KIIT University, Bhubaneswar, Odisha, India

Correspondence Address:
Dr. Nageswar Sahu
Department of Pathology, Kalinga Institute of Medical Sciences, KIIT University, Bhubaneswar, Odisha


Background: Lymphovascular invasion (LVI) is one of the important prognostic factors in carcinoma breast and its accurate diagnosis is important, particularly in node-negative cases for making the decision regarding aggressive adjuvant chemotherapy. Sometimes, it becomes very difficult to diagnose LVI on routine hematoxylin and eosin (H and E) stained tissue sections. Staining with endothelial immunomarkers makes the identification easy. Aim of the Study: This study aims to compare LVI detection on H and E and immunohistochemistry (IHC) stained sections and its correlation with axillary lymph node metastasis. Materials and Methods: A total of 45 cases of invasive breast carcinoma were studied. LVI was evaluated in routine H and E and IHC stained sections. The results were compared. The LVI status on IHC was correlated with axillary lymph node status. Results: The accuracy of H and E stained sections in detection of LVI was 75.6% with a false-positive and false-negative rate of 20% and 4.4%, respectively. There was no significant association between LVI on IHC and axillary lymph node status. Conclusions: The detection of LVI on routine H and E sections can be erroneous. IHC should be done in cases with confusing focus on routine H and E stained sections, particularly in node-negative cases.

How to cite this article:
Rahman K, Sahu N, Senapati U, Sahu SK. Importance of endothelial markers in detection of lymphovascular invasion in carcinoma breast and its correlation with axillary lymph node metastasis.Oncol J India 2020;4:23-27

How to cite this URL:
Rahman K, Sahu N, Senapati U, Sahu SK. Importance of endothelial markers in detection of lymphovascular invasion in carcinoma breast and its correlation with axillary lymph node metastasis. Oncol J India [serial online] 2020 [cited 2020 Sep 26 ];4:23-27
Available from: http://www.ojionline.org/text.asp?2020/4/1/23/282837

Full Text


Breast cancer is one of the leading causes of cancer death in females worldwide.[1],[2] Approximately 2.1 million new cases are diagnosed worldwide every year. In 2018, it was estimated that worldwide 627,000 women died of carcinoma breast, contributing approximately 15% of all cancer deaths among women. In India, it is the most common cancer among female.[2] In breast cancer management, it is important to decide and select the cases which will benefit from adjuvant chemotherapy. Prognostic factors, particularly tumor stage and axillary lymph node status, are one of the important deciding factors for adjuvant chemotherapy.[3] Lymphovascular invasion (LVI) is an independent poor prognostic factor and a strong predictor of lymph node metastasis.[4] Tumors with LVI but without lymph node metastasis, therefore, receive more intense therapy than cases without LVI in the same stage of disease. Even some authors recommend incorporation of LVI in breast cancer staging.[5]

Hematoxylin and eosin (H and E) stain is routinely used for the detection of LVI. The diagnosis of LVI is based on the presence of tumor emboli within an endothelial lined space, which may be either lymphatic vessel or blood vessel-like capillary or venous channel.[6],[7] Although many a times, it is easy to detect LVI in routine H and E stained sections; sometimes, it becomes difficult to accurately identify the vessel invasion leading to both false-positive and false-negative diagnosis. Factors that can interfere with the detection of LVI in routine H and E sections are peritumoral retraction cleft due to tissue shrinkage, small emboli, vessels completely filled up by the tumor emboli, or destruction of endothelial cells in the process of invasion.[7],[8] Furthermore, routine H and E fails to distinguish between lymph vessel invasion and blood vessel invasion in some situations.[6]

Many recent studies have given opinion that immunohistochemistry (IHC) using endothelial markers makes easier and more accurate identification of LVI in breast carcinoma. CD31 is a commonly used panendothelial marker for the identification of vascular channel.[9] Out of all the immunomarkers available for lymphatic endothelium, podoplanin (clone D2-40) is a sensitive, and relatively specific marker for lymphatic endothelium.[10] Hence, the combined use of CD31 and podoplanin is expected to make the detection of LVI more easy and accurate.

Therefore, we conducted the present study to detect LVI in routine H and E and by IHC using CD31 and podoplanin (D2-40) and to c orrelate LVI on IHC with axillary lymph node status.

 Materials And Methods

The present study was a prospective study conducted in the Department of Pathology, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha, India, over a period of 2 years from September 2017 to August 2019. The research proposal was approved by Institutional Ethics Committee (KIMS/KIIT/IEC/54/2017). The study included 45 cases of primary ductal carcinoma breast, undergoing lumpectomy, or mastectomy with axillary lymph node dissection. Cases with poor tissue fixation or with prior neoadjuvant treatment were excluded. We also did not include a case of mucinous carcinoma because of extensive extracellular mucin pool.

Grossing of the specimens were done as per guidelines of the National Cancer Institute, standardized management of breast specimen recommended by “pathology working group, breast cancer task force” with special emphasis on tumor size, multifocality, and lymph node sampling.[11] For the detection of LVI, multiple sections were taken from tumor periphery with peritumoral tissue, and in each case, one of the paraffin-embedded tissue sections was selected and subjected to IHC for comparison. Serial sections of the same paraffin-embedded tissue section were stained for H and E, CD31, and podoplanin, respectively. The antibodies and chemicals were obtained from Dako. The anti-human CD31, endothelial cell (clone JC70A165), and anti-human podoplanin (clone D2-40) monoclonal antibodies were used for CD31 and podoplanin, respectively. Immunohistochemical staining was done on formalin-fixed paraffin-embedded tissue sections on poly-L lysine coated slides using polymer two-step indirect method. The manufacturer's instructions were followed after standardization in our laboratory. Normal blood vessels and lymphatics in the sections were taken as internal positive control. Omission of primary antibody in a test case in each batch of staining was taken as a negative control. Routine histology reporting on H and E and detection of LVI on IHC was done by the first three authors. Tumor typing (as per the WHO classification) and grading (as per the Nottingham Modification of Bloom–Richardson grading system) were done on H and E stained section.[11],[12] LVI was searched in just outside the invasive tumor border in the selected H and E slide. Spaces lined by flat cell with a tumor emboli not fitting to the contour of the space or tumor cell nest attached to the wall were taken as LVI. The corresponding focus was analyzed on IHC for confirmation. Cases where LVI was not found on H and E were also searched for LVI on IHC slides. Any focus of LVI detected on IHC in these (H and E negative) cases, the corresponding focus was again reanalyzed on H and E slides to find the cause of false negativity.

Vessels were considered lymphatic if the lining endothelial cells were positive for podoplanin irrespective of CD31 status. Vessels with CD31 positive but podoplanin-negative endothelial cell lining were considered as blood vessels. Podoplanin shows diffuse stromal positivity in a few cases making the lymph vessel identification difficult.


A total of 45 cases of invasive ductal carcinoma were included in the study. All the cases were female with an age range of 34–76 years and a mean age of 55.6 years. Majority (71.1%) of the tumors fall into T2 category (2–5 cm) under tumor, node, metastasis classification. Most of the tumors belong to either Grade II (40%) or Grade III (42.2%). Axillary lymph node metastasis was detected in 51.1% of cases. On routine H and E stained sections, LVI was identified in 27 (60%) cases, but on IHC, only 20 (44.4%) cases showed the presence of LVI. Surprisingly, of these 20 cases, positive on IHC, few of them were negative for LVI on H and E sections [Table 1]. In all the cases with LVI detected on IHC, vessels were positive for podoplanin and of these 4 cases showed CD31 positivity. Hence, all the positive cases of LVI on IHC were lymph vessel invasion. We did not find any case of blood vessel invasion.{Table 1}

LVI detected by routine H and E and IHC were compared. In routine H and E evaluation, false-positive diagnosis was made in 9 (20%) cases and false-negative diagnosis in 2 (4.4%) cases [Table 1]. The sensitivity and specificity of H and E stained sections in detecting LVI were 90% and 64%, respectively, with an accuracy rate of 75.6% [Table 2].{Table 2}

We also analyzed the relationship between the presence of LVI (as per the IHC result) and axillary lymph node metastasis. Among all the cases with lymph node metastasis, 52.2% of cases did not show LVI on IHC, while 41% of the node-negative cases showed the presence of LVI, revealing no significant association (P = 0.64) between these two pathological parameters [Table 3].{Table 3}


We observed LVI in less number of cases using IHC as compared to routine H and E histology. Although Kenji et al. had a similar observation most of the other studies have a higher rate of detection of LVI using IHC for endothelial markers (CD31, CD34, Factor VIII, and podoplanin) as compared to routine histology.[1],[10],[13],[14],[15],[16],[17] This may be due to the stringent criteria employed by them in identifying LVI in routine histology or due to the different immunomarkers they have used.

False-positive and false-negative detection of LVI on routine H and E sections is a well-known observation. We analyzed the result of each case in H and E and IHC. In two cases, the LVI focus was missed on H and E but easily detected in the same tissue section using IHC. In one case, the vessel was irregular [Figure 1], and in the other cases, the LVI focus was too small and well away from the tumor periphery [Figure 2]. In nine cases, LVI was detected in routine H and E section, but the vessels in corresponding foci were negative for both CD31 and podoplanin on IHC indicating a high-false-positive rate of 20%. This high false-positive rate in the present study is mostly due to focal attachment of tumor cell nest to the wall in a retraction cleft, or irregular nonparallel contour of the adjacent stroma or the plump spindle cell nuclei of stromal fibroblast mimicking endothelial cells [Figure 3] and [Figure 4]. Hence, during the detection of LVI in routine H and E, stringent criteria should be applied to minimize false-positive diagnosis and hence preventing unnecessary treatment to decrease morbidity.{Figure 1}{Figure 2}{Figure 3}{Figure 4}

While analyzing the usefulness of H and E method in detecting LVI, sensitivity and specificity were found to be 90% and 64%, respectively, with an accuracy rate of 75.6% [Table 2]. Most of the previous such studies have low sensitivity but a higher specificity and variable accuracy rate for H and E method.[8],[10],[13],[17],[18] However, none of them found an accuracy rate of 90% or more thus highlighting the pitfalls of routine H and E method in detecting LVI in carcinoma breast. This highlights the importance of endothelial markers in detecting vessel invasion and thus reducing both false-positive diagnosis, which is very important, particularly in patients without any axillary lymph node metastasis to avoid unnecessary postsurgical treatment.

On IHC, we found only lymphatic vessel invasion. Blood vessel invasion was not seen. This may be due to the small sample size or preferred lymphatic route of spread in carcinoma breast. Abbasi et al., also in a study of more than 100 cases of carcinoma breast found lymphatic vessel invasion only.[13] Some other authors found blood vessel invasion though in less (<5.0%) number of cases.[8],[19] However, Marinho et al. found blood vessel invasion in more than 15% of cases.[10] This may be due to the larger sample size or the different endothelial markers used by them.

Lymph node involvement and presence of LVI are one of the important factors in deciding postsurgical treatment. Hence, the accurate detection of LVI is important, particularly in node-negative cases. We did not find significant association between LVI (IHC) and lymph node metastasis. Of 23 cases with lymph node metastasis only 11 (47.8%) cases showed the presence of LVI on IHC. This may be due to inadequate sampling of the peritumoral tissue. The analysis of entire tumor periphery may increase the chance of getting LVI in these cases. In addition, of 22 cases without lymph node metastasis LVI was detected on IHC in 9 (41%) cases. These cases with LVI will receive adjuvant chemotherapy thus highlighting the importance of accurate identification of vessel invasion. Dileep and Prasad also did not find significant association between the presence of LVI and lymph node metastasis.[15] However, many other studies demonstrated a significant association between these two histological parameters.[1],[8],[16],[18],[20],[21],[22]


Accurate identification of vessel invasion in carcinoma breast is of paramount importance particularly in node-negative cases. Many a times, it becomes very difficult to accurately identify LVI on routine H and E sections. Endothelial markers are helpful in correct identification of vessel invasion. Hence, we recommend IHC confirmation of any suspicious focus of LVI in carcinoma breast particularly in node-negative cases. Furthermore, IHC can differentiate between lymph vessel and blood vessel invasion. D2-40 shows diffuse stromal positivity in some cases making the lymph vessel identification difficult. The use of more endothelial markers will help to decide a more specific immune marker for lymphatic endothelial cell. The limitation of this present study is the small sample size and needs further evaluation with larger studies.


We would like to acknowledge our histotechnicians for preparing the slides.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Gujam FJ, Going JJ, Mohammed ZM, Orange C, Edwards J, McMillan DC. Immunohistochemical detection improves the prognostic value of lymphatic and blood vessel invasion in primary ductal breast cancer. BMC Cancer 2014;14:676.
2WHO. International Agency for Research and Cancer, in Latest Global Cancer Data; 2018.
3Song YJ, Shin SH, Cho JS, Park MH, Yoon JH, Jegal YJ. The role of lymphovascular invasion as a prognostic factor in patients with lymph node-positive operable invasive breast cancer. J Breast Cancer 2011;14:198-203.
4Lester SC, Kumar V, Abbas AK, Aster JC. The Breast. Pathologic Basis of Disease. 9th ed. New Delhi: Elsevier; 2015. p. 1066-8.
5Mohammed RA, Menon S, Martin SG, Green AR, Paish EC, Ellis IO. Prognostic significance of lymphatic invasion in lymph node-positive breast carcinoma: Findings from a large case series with long-term follow-up using immunohistochemical endothelial marker. Mod Pathol 2014;27:1568-77.
6Orbo A, Stalsberg H, Kunde D. Topographic criteria in the diagnosis of tumor emboli in intramammary lymphatics. Cancer 1990;66:972-7.
7Zaorsky NG, Patil N, Freedman GM, Tuluc M. Differentiating lymphovascular invasion from retraction artifact on histological specimen of breast carcinoma and their implications on prognosis. J Breast Cancer 2012;15:478-80.
8Arnaout-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.
9Van 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.
10Marinho VF, Sanches FS, Rocha GFS, Metze K, Gobbi H. D2-40, a novel lymphatic endothelial marker: Identification of lymphovascular invasion and relationship with axillary metastases in breast cancer. J Bras Patol Med Lab 2008;44:45-50.
11Rosai J. Breast. Rosai and Ackerman's Surgical Pathology. 10th ed. New Delhi: Elsevier; 2012. p. 1719-20, 2513-81.
12Sinn HP, Kreipe H. A brief overview of the WHO classification of breast tumors, 4th edition, focusing on issues and updates from the 3rd edition. Breast Care (Basel) 2013;8:149-54.
13Abbasi F, Abbasi A, Nabizadeh A, Esmaili A. Evaluation of lymphatic vessel invasion by immunohistochemistry in breast carcinoma. J Med Sci 2018;38:211-4.
14Norhisham NF, Chong CY, Safuan S. Peritumoral lymphatic vessel density and invasion detected with immunohistochemical marker D240 is strongly associated with distant metastasis in breast carcinoma. BMC Clin Pathol 2017;17:2.
15Dileep AP, Prasad PH. Use of immunomarkers D2-40 and CD31 in detection of lymphovascular invasion in breast carcinoma. J Med Sci Clin Res 2018;6:90-6.
16Lee JA, Bae JW, Woo SU, Kim H, Kim CH. D2-40, podoplanin, and CD31 as a prognostic predictor in invasive ductal carcinomas of the breast. J Breast Cancer 2011;14:104-11.
17Tezuka K, Onoda N, Takashima T, Takagaki K, Ishikawa T, Wakasa T, et al. Prognostic significance of lymphovascular invasion diagnosed by lymphatic endothelium immunostaining in breast cancer patients. Oncol Rep 2007;17:997-1003.
18Braun M, Flucke U, Debald M, Walgenbach-Bruenagel G, Walgenbach KJ, Höller T, et al. Detection of lymphovascular invasion in early breast cancer by D2-40 (podoplanin): A clinically useful predictor for axillary lymph node metastases. Breast Cancer Res Treat 2008;112:503-11.
19Mohammed RA, Martin SG, Gill MS, Green AR, Paish EC, Ellis IO. 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.
20Rakha EA, Martin S, Lee AH, Morgan D, Pharoah PD, Hodi Z, et al. The prognostic significance of lymphovascular invasion in invasive breast carcinoma. Cancer 2012;118:3670-80.
21Awamleh AA, Husban H, Rumman KA, Rabadi AA, Habahbeh S. The association between lymphovascular invasion and other prognostic indicators in operable breast cancer: Experience at King Hussein Medical Center. J Royal Med Serv 2016;23:11-6.
22Agarwal S, Singh A, Bagga PK. Immunohistochemical evaluation of lymphovascular invasion in carcinoma breast with CD34 and D2-40 and its correlation with other prognostic markers. Indian J Pathol Microbiol 2018;61:39-44.