Serum tryptase, mast cells positive to tryptase and microvascular density evaluation in early breast cancer patients: possible translational significance

Background Tryptase is a serine protease released from mast cells that plays a role in tumor angiogenesis. In this study we aimed to evaluate serum tryptase levels in 105 female early breast cancer patients before (STLBS) and after (STLAS) radical surgical resection, mast cell density positive to tryptase (MCDPT) and microvascular density (MVD). Methods STLBS and STLAS were assessed using the UniCAP Tryptase Fluoroenzyme immunoassay. Tumor sections were immunostained with a primary anti-tryptase antibody and an anti-CD-34 antibody by means of immunohistochemistry. Results The mean ± 1 standard deviation STLBS and STLAS was 7.18 ± 2.63 μg/L, and 5.13 ± 2.21 respectively and a significant difference between mean levels was found (p = 0.0001) by student t-test. A strong correlation between STLBS and MVD (r = 0.81, p = 0.0001); STLBS and MCDPT (r = 0.69, p = 0.003); and MCDPT and MVD (r = 0.77; p = 0.0001) was found. Conclusions Results demonstrated higher STLBS in breast cancer patients, indicating an involvement of MC tryptase in breast cancer angiogenesis. Therefore, serum tryptase levels may play a role as a novel surrogate angiogenic marker predictive of response to radical surgery in breast cancer patients. In this patients setting, it’s intriguing to hypothesize that tryptase inhibitors might be evaluated in clinical trials.


Background
Published data suggest that mast cells (MCs) have a dual role in the regulatory function between inflammatory and tumor cells. Interestingly, MCs induce tumor development and progression angiogenesis-mediated by means of the release of various angiogenic molecules such as Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor-2 (FGF-2), tryptase, chymase. On the other hand, MCs may induce apoptosis of malignant cells by means of the release of several cytokines such as interleukin-4 and Tumour Necrosis Factor [1][2][3][4][5].
Tryptase is an agonist of the proteinase-activated receptor-2 (PAR-2) in vascular endothelial cells and breast cancer cells that in turn stimulates their proliferation [24]. Tryptase also induces angiogenesis by release of stored angiogenic factors bound to the extracellular matrix [26][27][28][29]. Very little data have been published regarding the role of tissue MCs density positive to tryptase (MCDPT) in breast cancer (BC) angiogenesis and development [7,8,30]. Moreover, no reports are available about the role of serum tryptase in BC angiogenesis and as a circulating predictive surrogate marker.
Therefore, in the present study we aimed to evaluate serum tryptase levels in BC patients before (STLBS) and after (STLAS) radical surgical resection and MCDPT and microvascular density (MVD) in a series of tumor tissue from early BC patients to correlate each to other. The possible role of serum tryptase as predictive surrogate marker of radical surgery has been also evaluated. In this context tryptase inhibitors (gabexate and nafamostat mesylate) might be evaluated in adjuvant clinical trials as a new anti-angiogenic strategy.

Study populations
The clinicopathological features of the patients are summarized in the Table 1

Immunohistochemistry
The histological diagnosis was made on haematoxylineosin-stained slides and histopathological grading was performed according to the criteria described by Bloom and Richardson, as well, moderately and poorly differentiated state [32]. For the evaluation of MCDPT and MVD a three-layer biotin-avidin-peroxidase system was utilized [33]. Briefly, six-μm-thick serial sections of formalin-fixed and paraffin-embedded biopsy tumour were cut. Then, sections were microwaved at 500 W for 10 min, after which endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution. Adjacent sections were stained with human-specific monoclonal antibodies anti-tryptase (clone AA1; Dako, Glostrup, Denmark) diluted 1:100 for 1 h at room temperature and anti-CD34 (QB-END 10; Bio-Optica Milan, Italy) diluted 1:50 for 1 h at room temperature, respectively. The bound antibody was visualized using a biotinylated secondary antibody, avidin-biotin peroxidase complex and fast red. Nuclear counterstaining was performed with Gill's haematoxylin no. 2 (Polysciences, Warrington, PA, USA). The primary antibody was omitted in negative controls.

Morphometrical assay
An image analysis system (Quantimet500 Leica, Wetzlar, Germany) was utilized [33]. The five areas with higher immunostaining ('hot spots') were selected at low magnification and individual MCDPT ( Figure 1A) and MVD ( Figure 1B) were counted at x400 magnification (0.19 mm 2 area). The details of MCDPT and MVD were evaluated at x1000 magnification in oil ( Figure 1C and D, respectively).

Statistical analysis
MCDPT and MVD mean values ±1 standard deviations (s.d.) were evaluated by two independent observers (V.Z. and G.R.) for each tumor sample and in all series of sections. Correlations between STLBS, MCDPT, and MVD were calculated using Pearson's (r) analysis. The correlations between the above indexes and the clinicopathological features listed in Table 1 were analyzed by the Chi-square test. All statistical analyses were performed with the SPSS statistical software package (SPSS, Inc., Chicago, IL).
In this pilot study we have shown for the first time that STLBS strongly correlates with MCDPT and MVD in primary tumor tissue. Our data also demonstrates that STLAS significantly decrease in BC patients. Due to the release of tryptase from MCs, we suggest that MCDPT in primary BC tumor tissue represents the main source of serum tryptase. In our hypothesis, if primary tumor tissue is completely removed STLAS should decrease in one day due to their approximately 4-h long life-cycle. For these reasons, we detected STLAS 24 h before surgery to evaluate their possible role as a circulating surrogate marker suggesting the presence of tumor tissue, and again 24 h after treatment to confirm its decrease and, as a consequence, its possible expression of the absence of tumor tissue. We elaborate the background of our hypothesis based on previously published pilot data, which suggested an increase of MCDPT in primary tumor tissue. In these studies MCDPT was correlated with MVD, suggesting its role in BC angiogenesis [7,30]. Our data suggested an involvement of MCs and tryptase in BC angiogenesis. Interestingly, published studies already demonstrated an involvement of tissue MCDPT in other malignancies such as squamous carcinoma, gastrointestinal cancer, non-small cell lung cancer, melanoma, and endometrial carcinoma [9,11,12,18,19]. However, the above studies did not focus on the changes inSTLs before and after surgery, and no correlation between STLs, MCDPT and MVD was evaluated. It is remarkable that tryptase released from MCs is involved in tumor angiogenesis by several mechanisms. Firstly, tryptase stimulates the formation of vascular tubes in in vitro and in vivo experimental models; secondly, tryptase is an agonist of the PAR-2 in vascular endothelial cells that, in turn, induces angiogenesis [34]; thirdly, tryptase may stimulate the release of latent angiogenic factors bound to the extracellular matrix [21,35]. Overall, the above data suggest that tryptase may be a potential surrogate bio-marker of tumor angiogenesis which is able to predict response to surgical treatment.

Conclusions
If the primary source of tryptase production is no longer existing, after 24 h a significant reduction in STLs should be expected. If elevated STLs persist after surgery, this would suggest that residual tumor tissue remains after surgical resection or, alternatively, that unknown metastases are present. In this context, several tryptase inhibitors, such as gabexate or nafamostat mesilate, may be evaluated in future clinical trials as a new anti-tumor and antiangiogenic approach.