Pre-surgical level of von Willebrand factor as an evident indicator of breast cancer recurrence

1. Introduction

Breast cancer is one of the primary causes of cancer death in females. It is not a homogenous disease but it covers a group of diseases with specific clinical, histopathological and molecular features. According to the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2015 there can be distinguished different intrinsic subtypes of the disease, which include luminal A-like, luminal B-like, HER2-positive and basal-like. Because of the differences in biological features of breast cancers, they are additionally classified as well differentiated (low-grade), moderately differentiated (intermediate-grade) and poorly differentiated (high-grade) using the Elston-Ellis modification of Scarf-Bloom-Richardson grading system. Regarding the stage and molecular determinants, disease management can take different approaches [4].

It is well-established that neoplasm as well as a type of implemented anti-cancer therapy, including surgery procedure, chemotherapy are risk factors for hypercoagulability state. The prevalence of acquired thrombosis in cancer patients is 5-7-fold higher than in non-cancerous individuals [7, 18]. Thus, thrombosis is the second cause of death in hospitalized cancer patients. Importantly, 20% of patients with primary venous thromboembolism (VTE) constitute patients with cancer [7]. The participation in the cancer-related VTE development is attributed to platelets activation and its elevated count, as well as leukocyte count, haemoglobin, d-dimer, tissue factor (TF), soluble form of P-selectin (selectin expressed on platelets and leukocytes), factor VIII, or von Willebrand factor [3]. Additionally, activated platelets promote cancer development and dissemination and are considered as a ‘death mate’ of cancer. These phenomena occur via protection of cancer cells against the immune system and then enabling tumour cells survival in the circulation and by facilitating their adhesion to the endothelial cells (ECs) mediated by selectins [14, 18]. The cancer-dependent thrombocytosis is associated with shortened survival and poor prognosis [26].

Selectins belong to a family of adhesion molecules that mediate interactions between tumour cells, platelets and endothelial cells [14, 21]. P-selectin is a cell adhesion molecule that can be found in the membranes of alpha granules of platelets and the Weibel-Palade bodies of endothelial cells granules. Circulating P-selectin (soluble form of P-selectin) is shedding from damaged or activated platelets or endothelial cells as well as by proteolytic cleavage of the transmembrane protein [3, 15]. Soluble P-selectin is elevated in breast cancer females, further confirming the platelet activation in cancer progression [25]. It has been found that P-selectin deficiency diminishes number of metastasis in a xenographic model of human BrC cells, but it insufficiently abolish them what may suggest a participation of other adhesion molecules in cancer progression [13]. In respect to E-selectin (selectin expressed on endothelial cells) is an adhesion molecule, which is not expressed on the surface of endothelium in quiescent state. However it appears in response to cytokines such as tumour necrosis factor α (TNF- α ) or interleukin 1 β (IL1 β ) and is involved in cancer-associated inflammation as well as in neoangiogenesis, cancer cell trafficking and metastatic spread of cancer cells. An increase E-selectin level may be a prognostic marker in the course of cancer, but its effect is largely unknown [28].

Interestingly, the principal platelet adhesion ligand, von Willebrand factor, also is involved in cancer growth and progression [1]. In the case of endothelial injury, platelets immediately co-operate with the vessel wall in a glycoprotein (GP) Ib-V-IX-Von Willebrand Factor (VWF)-linked mechanism leading to the platelet adhesion and then aggregation [14]. Several studies suggest that VWF is also associated with angiogenesis, smooth muscle proliferation, and creation of secondary tumour sites [9]. An elevated concentration of plasma VWF is associated with the generation of cancer-related thrombosis, as well as with the degree of malignancy, the rate of metastasis and patients future outcomes [17, 27]. However, an adequate role of VWF in this processes remains unclear and require further elucidation. Since, there are several studies pointing out that VWF exerts antitumor properties, mainly by suppressing angiogenesis and pro-apoptotic effect [16, 20, 24].

The activation of platelets and coagulation process by cancer cells is not just a risk factor for developing thrombosis, but also way that tumour cells promote angiogenesis and metastasis. The inflammatory state in cancer is enhanced by platelets which activate endothelial cells and recruit leukocytes to primary and secondary tumour sites. Thus, the aim of this study was to assess the concentration of VWF, soluble forms of P-selectin and E-selectin in blood of breast cancer patients and their mutual connections in the course of the disease. We also aimed to evaluate of VWF, sP-selectin and sE-selectin as indicators of future prognosis.

2. Materials and methods

4. Discussion

In the present study the concentrations of VWF, sE-selectin and sP-selectin as well as VWF/sE-selectin and sP-selectin/sE-selectin ratios were analysed in patients with IBrC to determine whether measured parameters were useful indicators for disease relapse prediction. The results of the present study suggest that the VWF antigen and VWF/sE-selectin ratio are potential biomarkers for future prognosis in IBrC patients. We believe that VWF level is a more useful biomarker than other analysed proteins.

In physiological conditions von Willebrand factor plays a key role in the initiation of platelets adhesion and further aggregation as well as in the secondary haemostasis by binding factor VIII prolonging its half-life in the blood [16]. However its contribution to various processes, such as thrombosis, inflammation, angiogenesis and eventually cancer metastasis cannot be omitted [9]. The VWF disruption in the course of malignant diseases, including leukemias, colorectal cancer, leiomyosarcoma, breast cancer and prostate cancer has been reported [8, 17, 27]. In our study, regardless of analysed subgroup of IBrC the VWF concentration were higher than the normal range (50–150 mU/mL) reported in the general population. Interestingly, a positive correlation between the concentration of VWF and a tumour grade in IBrC subjects was noted, which is in line with Pepin et al. study [17]. The precise mechanism between elevated VWF levels and neoplastic diseases is quite complicated to define, since VWF is an acute phase protein and an endothelial dysfunction marker and its concentration depends on stimulation of sympathetic nervous system, infection, aging process, even a blood type [16, 20]. However, extremely high level of VWF (over 600 mU/mL) as well as a higher vWF/sE-sel ratio than 17.5 were linked to better survival rates in breast cancer patients. The results refer to the whole study group and to women without nodal involvement as well as with luminal A and luminal B HER2- tumours. This observation is consistent with previous study, which demonstrates anti-metastatic role of VWF in experimental mouse model. Terraube et al. observed an increased number of pulmonary metastasis in the absence of VWF. Authors demonstrated that VWF is important agent in protection against tumour cell spread in vivo, interestingly the mechanism of metastasis inhibition and tumour cells death induced by VWF is independent of its function in coagulation [24]. Tigges et al. claim that endothelial VWF deficiency can promote angiogenesis [23]. Also, Stark et al. shown that the absence of VWF enhances angiogenesis. Authors found that inhibition of VWF expression by short interfering RNA (siRNA) in vitro led to in increased integrity of the vessel network via an increased vascular endothelial growth factor receptor type 2- dependent (VEGFR-2) proliferation and migration of endothelial cells. They also observed that neo-angiogenesis and vascularization are enhanced in the VWF-deficient mouse. The capillary tube network was markedly greater in VWF-deficient cells, pointing out that both extracellular and intracellular VWF controls angiogenesis. Stark et al. speculate that elevated VWF levels, as reported in many diseases, may suppress angiogenesis [20]. However, Pepin et al. suggest that high VWF levels are correlated with cancer dissemination [17]. Moreover, Obermeier et al. reported that high VWF levels were independently associated with worse overall survival [10]. Despite of lack of scientific consensus in respect to VWF role in neoplastic diseases; in our study at the end of the observation period of 40 months, 11 of 13 events occured in patients with VWF value below 600 mU/mL with recurrence rate of 25%, but only two events occurred in subject with VWF values above the 600 mU/mL (5%; P = 0.0028). We postulate that should be established a cut-off value for VWF concentration to consider the degree of its elevation, since all cancer patients present higher VWF concentration than physiological values. Notable, among patients with a high VWF we can distinguish those with better and worse future prognosis. Perhaps, pro-metastatic potential depends on VWF concentration. Our study demonstrates a strong enough that VWF could be considered as an accurate and specific biomarker of breast cancer relapse. In this context, we speculate that our results demonstrate of major practical importance. Appling an easy, commercially available immunoassay kits developed to measure VWF concentration, we were able to identify patients with breast cancer in whom the risk of disease recurrence is considerably elevated, regardless of analysed subgroup. These findings could have diagnostic, clinical, prognostic implications in the future. We would recommend analysis of VWF levels in subsequent studies.

Furthermore, according to statistical analysis we observed a higher concentration of sP-selectin in hypertensive patients than non-hypertensive ones as well as a sP-selectin level was negatively associated with the age in the group of IBrC patients without nodal involvement. It is worth a mention that platelet P-selectin has been acknowledged a marker for prediction of thrombosis and angiogenesis in breast cancer. P-selectin not only mediates the adhesion of leukocytes, platelets, and cancer cells during inflammatory processes and thrombosis but also contributes to cancer growth and metastasis. The P-selectin mediates tumour cells dissemination out of the tumour microenvironment by supporting arrest of cancer cells in the circulation and extravasation at distant sites [12]. This is consistent with our results, where lymph node-negative patients shown a shorter recurrence-free survival with higher concentrations of sP-selectin. Furthermore these results suggest activation of platelets even in patients are at early stage of the disease. Based on previous study we suggest, that in the group of IBrC patients with elevated sP-selectin, the higher risk of disease progression or recurrence is associated with massive platelet activation, but also independently with thrombosis development, since elevated level of sP-selectin may strongly predict venous thromboembolic event (VTE) in neoplastic patients [5]. Qi et al. demonstrate that platelets are actively penetrate to solid tumours, including insulinoma and malignant melanoma, via a P-selectin-related pathways, where they release VEGF, and other pro-angiogenic mediators, thereby leading to formation new capillary network and supporting tumour growth [6]. Undoubtedly, elevated level of sP-selectin is negative prognostic indicator. Since, according to our study in patients with sP-selectin above 250 ng/mL, the recurrence rate was 30.43%, whereas IBrC patients with lower sP-selectin 250 ng/mL had a cumulative probability of merely 2.5% of disease recurrence.

We observed a higher concentration of sE-selectin in post-menopausal subjects in respect to pre-menopausal IBrC cases. Also in patients aged 55 or more and those who were overweight and obese there was a tendency towards a higher concentration of sE-selectin. Ragab et al. observed elevated serum levels of soluble form of E-selectin in patients with breast cancer compared to benign group and compared to their healthy counterparts [11]. Furthermore Kang et al. reported a positive association between a sE-selectin concentration with tumour diameter, grade, stage, molecular subtype and worse survival in breast cancer [22]. During metastatic spread of cancer cells into distant tissues, inflamed endothelium expresses different adhesion molecules, including E-selectin, which most important role is to mediate rolling and vascular adhesion of breast cancer cells and leukocytes [2]. Furthermore circulating form of E-selectin potentiates adhesion and migration of these cells independently from vascular endothelium and facilitate tumour progression and metastasis [22]. The involvement of E-selectin on tumour progression is still unknown. Most likely, the role of E-selectin in tumour cells dissemination is associated with attachment and diapedesis of tumour cells through the vessel wall [2, 22]. Furthermore, we noticed that a higher concentration of sE-selectin at the time of diagnosis were not linked with worse clinical outcome. Our findings deviate from the view of using of sE-selectin as a biomarker for early detection of metastasis for several types of cancer. The applicable value of sE-selectin as a single biomarker may require more accurate consideration, as its concentration may be influenced different factors, hence sE-selectin show a limited practicality in a clinical setting. Thus we suggest to juxtaposition of sE-selectin with second endothelial activity/function biomarker- VWF, since both markers are sensitive on endothelial cells stimulation. In our study the VWF/sE-selectin ratio was significantly higher in patients with grade 3 than in those cases with grade 1 and 2 tumours as well as it correlated positively with tumour grading and negatively with expression of oestrogen receptor. There was also a tendency for higher VWF/sE-selectin ratio in premenopausal women and those who were younger than 55 years old. Furthermore we investigated the prognostic value of VWF/sE-selectin ratio – lower ratio than 17.5 was associated with worse survival rate in the whole study group, as well as in a group of patients without nodal involvement and in a group of patients with luminal A or B HER2- subtype of breast cancer (recurrence rate: 21.43%, 19.35%, 26.67%, respectively).

The present study has several limitations, including a short observation period and quite small sample size. Breast cancer patients have been in peri-menopausal period, which is associated with age-related comorbidities including weight gain, elevation in blood pressure or hormone imbalance, which may impact on the VWF, sE-selectin and P-selectin levels.

Our study demonstrates that VWF could be considered as an accurate and specific biomarker of breast cancer relapse, regardless of analysed subgroup. However, we claim that should be established a cut-off value for VWF concentration to consider the degree of its elevation. We propose, that the sP-selectin concentration over 250 ng/mL is connected with the higher risk of disease progression. A soluble form of E-selectin does not present any essential value in malignancy disease prediction. We believe that VWF/sE-selectin ratio is a more suitable indicator of tumour progression and spread than a sE-selectin as a single parameter.