Correlation between serum tryptase, mast cells positive to tryptase and microvascular density in colo-rectal cancer patients: possible biological-clinical significance

Abstract

Background: Tryptase is a serin protease stored and released from mast cells (MCs) that plays a role in tumour angiogenesis. In this study we aimed to evaluate serum tryptase levels in colo-rectal cancer (CRC) patients before (STLBS) and after (STLAS) radical surgical resection. We also evaluated mast cell density positive to tryptase (MCDPT) and microvascular density (MVD) in primary tumour tissue.

Methods: A series of 61 patients with stage B and C CRC (according to the Astler and Coller staging system) were selected. Serum blood samples were collected from patients one day before and one day after surgery. Tryptase levels were measured using the UniCAP Tryptase Fluoroenzymeimmunoassay (Pharmacia, Uppsala, Sweden). Tumour sections were immunostained with a primary anti-tryptase antibody (clone AA1; Dako, Glostrup, Denmark) and an anti CD-34 antibody (QB-END 10; Bio-Optica Milan, Italy) by means of immunohistochemistry and then evaluated by image analysis methods.

Results: The mean ± s.d. STLBS and STLAS was 5.63±2.61 µg/L, and 3.39±1.47 µg/L respectively and a significant difference between mean levels was found: p = 0.000 by t-test. The mean ± s.d. of MCDPT and MVD was 8.13±3.28 and 29.16±7.39 respectively. A strong correlation between STLBS and MVD (r = 0.83, p = 0.000); STLBS and MCDPT (r = 0.60, p = 0.003); and MCDPT and MVD (r = 0.73; p = 0.001) was found.

Conclusion: Results demonstrated higher STLBS in CRC patients, indicating an involvement of MC tryptase in CRC angiogenesis. Data also indicated lower STLAS, suggesting the release of tryptase from tumour-infiltrating MCs. Serum tryptase levels may therefore play a role as a novel bio-marker predictive of response to radical surgery. In this context tryptase inhibitors such as Gabexate and Nafamostat Mesilate might be evaluated in adjuvant clinical trials as a new anti-angiogenic approach.

Figure 1. Colon cancer sections immunostained for the evaluation of MCDPT and MVD at x400 magnification, high MCDPT and MVD respectively.

In A a primary anti-tryptase antibody has been employed. Arrows indicate red mast cells positive to tryptase. In B a primary anti CD-34 antibody has been employed. Arrows indicate red positive microvessels.

Figure 2. Colon cancer sections immunostained for the detail evaluation of MCDPT and MVD at x1000 magnification in oil respectively.

In A a primary anti-tryptase antibody has been employed. The big arrows indicate single red mast cells positive to tryptase. The small arrow indicates the degranulation front of the mast cell forming a microvessel. In B a primary anti CD-34 antibody has been employed. The big arrow indicates a red positive microvessel. The small arrow indicates the nucleus of the endothelial cell. Note the lumen of the microvessel.

Figure 3. Adjacent normal mucosa section and colon cancer section immunostained for the evaluation of MCDPT and VEGF expression at x400 magnification respectively.

In A a primary anti-tryptase antibody has been employed. Arrow indicates a single and unique red mast cells positive to tryptase in the observed field. In B a primary anti-VEGF antibody has been employed. Many red immunostained cancer cells. Big arrows indicate single red cytoplasmic cancer cells positive to VEGF.

Figure 4. Differences between STLBS (red line) and STLAS (blu line).

Figure 5. Correlation analysis between MVD and STLBS.

Figure 6. Correlation analysis between MCDPT and STLBS.

Figure 7. Correlation analysis between MVD and MCDPT.