In this study, we identified vessel size as a marker of poor prognosis in the ER + subset of breast cancer. Key findings for this claim are the identification of significant associations between this marker and poor prognosis in multivariable analyses of two patient series, one consisting of 108 cases analyzed as whole sections and the other consisting of 267 TMA cores of ER + breast cancer samples.
Some strengths of this study are that the prognostic metric was scored using an automated method increasing objectivity, and also that associations were detected following a simple median-based dichotomization. One limitation, to be addressed and considered in future validation studies, is that the design for the study prevents analyses from clarifying if the survival associations are related to intrinsic tumor aggressiveness or response to subsequently administrated treatment.
Our study was designed to examine the prognostic value of a-SMA perivascular coverage, vessel density and vessel size on breast cancer specific survival. The initial data showed high inter-case heterogeneity of all these three metrics (Fig. 1a). Correlation analyses pointed out a positive correlation between vessel median size and fraction of a-SMA covered vessels, and a negative correlation between vessel median size and vessel density (Fig. 1b). None of our tissue metrics showed any associations with clinico-pathologic characteristics in either of the two series (Suppl. Tables 2–4, Suppl. Tables 6–12). Although perivascular a-SMA coverage has been observed as a prognostic factor in different studies and was associated with poor prognosis in some of them (31–34), in our breast cancer study this metric showed no association with survival (Suppl. Figure 1).
To the best of our knowledge, the only earlier study reporting vessel size as a potential prognostic marker performed on breast cancer patients is by Mikalsen et al. (40). Our observation is in concordance with their finding that large vessels are associated with shorter breast cancer specific survival. In the study of Mikalsen et al., the authors also focused their attention on the vessel shape complexity as an important factor of survival (40), which was not examined in our present study. To note, our study demonstrated the independent prognostic value of median vessel size when adjusting for age, tumor size, histological grade, HER2 status, progesterone receptor status, and lymph node status in multivariable Cox analysis, not previously shown in ER + breast cancer subtype (Table 3).
Vessel density failed to provide independent prognostic value in our study (Suppl. Figure 1). This vascular feature has been analyzed in earlier studies, and have provided divergent results (12, 14, 43, 44, 15–20, 41, 42), (Suppl. Figure 1). Tentative reasons for these discrepancies include scoring of different tumor sub-regions, and variations in methods for vessel scoring (45). Furthermore, some studies pointed out that vessels with certain qualities can have favorable effects on prognosis (46). This could explain why our study, among the few others, failed to replicate high microvessel density being a significant factor of poor prognosis, having vessel number taken without considering further their morphological features and also the molecular landscape of the involved endothelium, which has been previously known to have a clinical significance (6, 8, 46–48). Notably, more consistent signals have been obtained when the density of proliferating vessels has been analyzed (15–20).
In different clinico-pathologic subsets, the impact of vessel diameter on survival in the whole section cohort was significant only in ER/PR + subsets, breast cancers with lymph node metastases, and Lum B/HER2- molecular subtype (Fig. 3, Supp. Table 5). It has been known that endothelial cells contain estrogen receptors (49, 50) and that estrogen fulfills its effects as a vascular protector in premenopausal women, aside from other mechanisms, through the reduction of peripheral vascular resistance by increasing vessels’ lumen size (51). Moreover, it has been reported that ER expression in breast cancer will correlate with higher levels of available estrogen hormone in the breast tissue, which can also be correlated with higher estrogen hormone delivery induced by the higher tissue vascularization (52). Along with this statement, in the study by Lloyd et al., the authors hypothesized that the blood flow arrangements can be the cause of breast cancer cell heterogeneity and that defining vessel characteristics could help to predict ER positivity pattern. In this study, the authors reported a strong positive correlation between the vessel size and the positive ER status in breast cancer, observing the mean vessel diameter of ER positive tumors being around twice of the mean vessel size in ER negative tumors (52). Their finding is providing a possible explanation for why our large vessel prognostic signal was enriched particularly in the ER + tumors. Nevertheless, we failed to observe an association between vessel size and ER status in our study (Suppl. Table 3). Lloyd et al. also recorded that vessel density was not correlated with the ER status or disease progression (52), which is in concordance with our present findings (Suppl. Table 2).
Additional questions that arise from the biological perspective are by which mechanisms large vessels could affect cancer biology, cancer progression, metastasis and eventually survival. The aberrant vessel anatomy, along with the presence of big and distorted vessels, is known to influence dysfunctional blood flow, perpetuate extravasation of cancer cells and facilitate metastatic processes, consequentially having a negative effect on overall survival (11). Angiogenesis has been recognized as an essential piece of the puzzle in the process of tumor metastasis (53). Although we did not find a correlation between vessel median size and vessel density with the occurrence of lymph node metastasis, previous studies are suggesting an association between vessel size and higher metastatic potential (40, 54–57). In addition, it has been noted that an increase in the mean vessel diameter is associated with tumor angiogenesis and with a larger tumor size (58).
An additional, specific tumor vessel morphology has been described by Senchukova and Kiselevsky in 2014 (59) when they reported the existence of so-called “cavitary structures“ (CS), which are stromal structures lined with endothelium and connected with the rest of the tumor vasculature. In our study, we indeed recorded similar structures in patients belonging to the “high vessel median size” group, as shown in Fig. 4a. Additional studies conducted on these structures, defined specific molecular signatures of tumor tissues containing CS, such as high levels of nitric oxide synthase (iNOS), increased synthesis of thrombospondin 4 and high levels of matrix metalloproteinases (Senchukova et al., 2015). In the study from 2015, Senchukova et al. reported two different types of “cavitary structures”, cavitary structure type 1 and type 2 (CS-1, CS-2) (54). The authors provided evidence that the specific type of these “cavitary structures” (CS-1) is being associated with lymphovascular invasion, presence of tumor emboli in vessels, and clinically evident metastasis in gastric cancer (54) and breast cancer (56). Moreover, they reported that the formation of CS-1 was associated with high density of CD68 positive cells in the surrounding stroma and that high density of CD20 positive cells was associated with the formation of CS-2 type (54). This can suggest biological mechanisms and signaling pathways that might be examined in the future. Further studies investigating the involvement of stromal and immune cells in the formation of CS and big size vessels might prove to be beneficial in detecting pathways and mechanisms involved in angiogenesis, metastasis, and tumor progression and which could be potentially exploited as druggable targets or biomarkers of survival and prognosis.