Abstract P6-02-04: TMEM (Tumor MicroEnvironment of Metastasis) in human breast cancer is a blood vessel associated intravasation microenvironment unrelated to lymphatics
Authors: Ginter PS , Robinson BD , D'Alfonso TM , Oktay MH , Gertler FB , Rohan TE , Condeelis JS , Jones JG 
Published: August 2014

Abstract
In breast cancer, both lymph node and distant metastasis represent dissemination of tumor cells from a primary site, but the mechanism of spread and the subsequent risk of mortality may not be the same. Historically, lymphatic spread has been documented both descriptively, as presence or absence of lymphovascular invasion (LVI), and as a formal part of TNM staging. Until recently, however, there has been no way to directly assess the risk of hematogenous dissemination by the primary tumor.
Observations from multiphoton-based intravital imaging of rodent models of breast cancer and the analysis of Mena function in tumor cells in vivo have characterized an intravasation microenvironment (ME) involved in the systemic dissemination of tumor cells from primary breast tumors. We have identified the corresponding structure in FFPE tissue and called it TMEM (Tumor MicroEnvironment of Metastasis). This microanatomic landmark is defined as the direct apposition of a Mena-overexpressing intravasation competent carcinoma cell, a perivascular macrophage, and an endothelial cell. In a case control study of 30 case-control pairs, where each matched pair differed only in their metastatic status – non-metastatic vs. metastatic – we found that the density of TMEM was significantly associated with development of systemic metastasis (p = 0.00006).
The relationship of hematogenous- and lymphatic-mediated tumor cell spread is not understood. Using the previously described cohort in which we showed that TMEM was associated with metastasis, the purpose of this study was to 1) assess intratumoral lymphatic density, 2) determine if TMEM- lymphatic structures associated with lymphatics exist, and 3) determine if TMEM- lymphatic structures correlate with systemic metastatic risk. Cases were stained with a triple immunostain identical to that used in our earlier study except that D2-40 (a lymphatic marker) was used, rather than CD31 (a blood vessel marker). The marker for macrophages (CD68) and invasive tumor cells (Mena) remained the same. Two pathologists, blinded to outcome, evaluated the presence or absence of intratumoral lymphatics and quantitated the number of TMEM-lymphatic structures per 10 high power (400x) fields in areas of highest intratumoral lymphatic density. A TMEM-lymphatic structure was defined as the direct apposition of a lymphatic (D2-40) endothelial cell with a macrophage and invasive tumor cell.
Intratumoral lymphatics were absent in a majority of tumors in each of the 2 groups (18 of 30 non-metastatic, 16 of 30 metastatic; p = 0.6). TMEM-lymphatic structures were rare and were equally present in the 2 groups (3 metastatic and 3 non-metastatic cases). Using the Wilcoxon (paired) signed-rank test, we found no significant difference in the density of these structures between the two groups (p = 0.4). Furthermore, TMEM-lymphatic structures did not correlate with the presence of lymph node metastases (p = 0.8). We conclude that lymphatic vessels do not participate in the TMEM assembly that has been associated with hematogenous metastasis. TMEM density assessment reflects a hematogenous intravasation ME and offers a novel approach to the assessment of metastatic risk.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-04.