Tumor angiogenesis is a highly complex process that involves accurate communication of tumor cells with their host organs or tissues, which is controlled by the interplay of a wide range of factors 18. Angiogenic factors encourage the formation and development of blood vessels by cancer cells to expand tumors 19. Several studies have shown that angiogenic factors have a significant role in tumor growth and expansion. According to the outcomes of immunohistochemical analyses, the members of VEGF family and their receptors are expressed in almost half of human cancers. Moreover, a significant association between the expression of VEGF and prognosis has been described in colorectal, breast, lung, head and neck squamous cell carcinomas, Kaposi sarcoma and malignant mesothelioma. These researches have also demonstrated that the levels of angiogenic factors in a tissue indicate the aggressiveness of tumor cells, and thus have predictive value in recognizing patients with poor prognosis who are at high risk 20.
Several studies have shown that overexpression of angiogenic factors such as PD-ECGF, bFGF, TGF-β, angiogenin, and COX-2 in different cancers are correlated with the advanced tumor stage and decrease patient survival 21. Aside from the VEGF family, FGFs are also known as a family of potent angiogenic motivators associated with the risk of breast cancer 22. While FGF-1 is known as an acidic polypeptide, FGF-2 is a bFGF polypeptide and plays a pivotal role in the stimulated proliferation and differentiation of endothelial cells. In addition, a significant association between high serum or urine levels of bFGF and progressive disease in patients with different types of cancers has been reported 22,23.
TGF-β is secreted by both of normal and cancerous cells. Depending on the stage of breast cancer development and progression, it can act as either a pro- or anti-oncogenic protein. TGF-β is a highly oncogenic factor in the late stage, aggressive and metastatic breast cancers 24. According to recent studies, the high expression of angiogenesis-related proteins is associated with adverse clinicopathological parameters in the early-stage breast cancer patients 25.
Radiation can cause damage to the microenvironment of both cancerous and normal cells (like endothelial cells). There are conflicting reports on the consequences of radiation. Some studies indicate that radiation may enhance tumor invasiveness and metastasis. These observations may be explained by the fact that cancer cells are destroyed or damaged by radiation, thereby secreting a variety of soluble factors that promote angiogenesis and improve migration and invasion of cancer cells 26,27. Destruction of epithelial cells by radiation depends on the beam dose. It has been generally reported that higher doses in the range of 2–15 Gy have an anti-angiogenic effect, while lower doses about 0.5–0.8 Gy appear to be pro-angiogenic 28,29. However, it can be more complicated than it seems. For instance, it has been reported that a single high dose (20 Gy) of radiation to the mammary gland decreases the local vessel density in a mouse model of breast cancer relapse, after injection of tumor cells 30. Radiation has been reported to alter the expression of cytokines in the wound fluid 15. Furthermore, IORT changes the expression of miRNA223, thereby reducing EGF expression and EGF receptor activation, a cascade that normally inhibits the growth of breast cancer cells and decreases the risk of local tumor recurrence in mice models 31.
In the present research, the concentration of EGF in both IORT and Non-IORT groups was increased after surgery. Our results also indicated that IORT decreases the DLL4 level. In addition to VEGF, EGF level was also increased after IORT intervention. Interestingly, IORT and LN involvement were influential on the EGF level. EGF was indeed a significant LN involvement predictor. Moreover, IORT, vascular invasion and LN involvement have significant impacts on the serum levels of TGF-β and EGF. The FGF level in WF was influenced by IORT, LN involvement and stage, while the size of tumor and the subsequent TNM stage affected the difference between the pre- and post-surgery levels of DLL4 and VEGF expression. DLL4 is a critical factor in vascular maturation and tumor angiogenesis and plays a key role in VEGF signaling 32. A recent study has shown that VEGF secretion by tumor cells is essential for tumor development in the early-stage of breast tumors 33.
An attractive finding obtained from the ROC analysis was that DLL4 and EGF levels can be used to differentiate the late stages of disease from early stages, LN involvement from free LN, and high tumor size from low tumor size. On the other hand, these two biomarkers could predict the end-TNM-stage. According to the results of previous studies, the serum level of TGF-β is an early marker for predicting fibrosis after surgery and before radiotherapy. The serum levels of TGF-β in patients who had undergone IORT after surgery were significantly higher than those of the patients that had only undergone breast-conserving cancer surgery, suggesting that this alteration in the TGF-β level was the outcome of IORT 34.
Keegan et al. have demonstrated that young patients with breast cancer are associated with more advanced stages, such as higher T and N stages 35. On the contrary, our Cox risk regression analyses showed that age more than 40 years is a risk factor for the overall survival. In addition, tumor grade, estrogen receptor (ER) and progesterone receptor (PR) were other independent risk factors for a poor prognosis of breast cancer and had negative impacts on the overall and recurrence-free survival of patients. Another interesting finding was the effect of IORT on the overall and recurrence-free survival.
We demonstrated that treatment with IORT reduces the risk of death and the recurrence rate in comparison to the Non-IORT group. Furthermore, several randomized trials have demonstrated excellent early tumor control, survival, and cosmetic outcomes following IORT in the breast cancer patients 36. Vaidya et al. have performed a prospective randomized study on the IORT treatment versus the whole-breast radiotherapy based on a four-year dataset. They demonstrated a local recurrence rate of 1.2% in the IORT group versus 0.95% in the external beam radiotherapy group 16. In another research, they have reported a 5-year risk for local recurrence in the conserved breast equal to 3.3% for targeted intraoperative radiotherapy (TARGIT) versus a value of 1.3% for the adjuvant whole-breast external beam radiotherapy (EBRT). However, the mortality due to breast cancer in the TARGIT vs EBRT groups were the same 37.
In our study, the mean follow-up duration was 24 hours after treatment, while a follow-up of 48 or 72 hours may lead to more accurate results on the effect of IORT on the angiogenic factors. However, patients have been following up for more than 5 years now. Overally, IORT was found to offer a potential survival advantage and it can help to reduce recurrence.