Prognostic Significance of Mast Cell Number and Microvascular Density for the Survival of Patients With Primary Colorectal Cancer

Maya Gulubova; Tatyana Vlaykova

Disclosures

J Gastroenterol Hepatol. 2009;24(7):1265-1275. 

In This Article

Methods

Patients

This study involved the analysis of 106 patients, who had undergone tumor resections for adenocarcinoma of the colon and rectum between January 2001 and December 2003 at the University Hospital, Medical Faculty, Trakia University, Stara Zagora.

The patient population consisted of 43 men and 63 women, aged between 37.8 and 82.3 years (mean of 62.8 ± 10.3, median of 63.3). Forty-nine (46%) of the patients suffered from rectal and 57 (54%) from colon cancer. Tumor grading and staging was performed according to the tumor-node-metastasis (AJCC Cancer Staging schemes) classification. Ten of the patients had tumors in stage I, 68 in stage II, 16 in stage III and 12 patients had tumors in stage IV. Patients did not receive chemotherapy or radiation therapy before surgery. All had primary tumor resection with regional lymph node dissection. Informed consent was obtained from all patients. The operations were carried out according to the accepted protocols in Bulgaria for surgical interventions and obtaining of human biopsy materials.

The patients were followed up until 30 June 2006, when 49 (46%) of them were alive. The survival of all patients after the operation varied from 1.1 month to 66.6 months with a median of 32.2 months. Half of the patients received adjuvant standard monotherapy of 5-FU/folic acid (5-FU/FA) or polychemotherapy consisting of 5-FU/FA plus camptothecin or mitomycin C or oxaliplatin.

Immunohistochemistry

Tumor specimens obtained were fixed in 10% buffered formalin and embedded in paraffin. Histological grading was performed on hematoxylin-eosin stained sections.

The histochemical staining of mast cells was with toluidine blue (TB) and was performed by the standard histochemical method: slides were incubated with 0.1% toluidine blue for 3 min and then rinsed with distillated water.

Immunohistochemical staining for CD31 and mast cell tryptase was performed using the streptavidin-biotin-peroxidase complex technique on formalin-fixed and paraffin-embedded tumor sections as described earlier.[22] Briefly, mouse monoclonal antibody to human CD31, clone Gi18 (Bender MedSystems Diagnostics, Vienna, Austria) diluted 1:300 in 0.3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) and mouse monoclonal antibody to human mast cell tryptase, clone AA1 (M7052; Dako, Glostrup, Denmark) in a dilution 1:50 were used. Sections 5 μm thick were dewaxed twice in xylene at 56°C for 1 h and were rehydrated in ethanol. Then the sections were soaked in 10% sucrose in distilled water overnight. Antigen retrieval was performed in alkaline solution, pH 10 (0.1 mol/L Tris-NaOH) for 1 min at 750 W, followed by 5 min at 500 W in a microwave oven. After cooling for 15 min, the slides were rinsed in PBS, pH 7.4, incubated in 1.2% hydrogen peroxide in methanol for 30 min, and rinsed in PBS, pH 7.4, for 15 min. The sections were then blocked for 30 min with normal goat serum (Dako). After incubation with the primary mouse antihuman antibodies overnight at 4°C, slides were washed three times for 5 min each in PBS, pH 7.4, and incubated with a secondary antimouse biotinylated antibody (Dako ready-to-use LSAB®2 System, horseradish peroxidase [HRP] K0675) for 4 h at room temperature, and subsequently with the streptavidin-HRP complex (Dako ready-to-use LSAB®2 System, HRP K0675) for 4 h at room temperature, rinsed in PBS, pH 7.4, and then in 0.05 mol/L Tris-HCl buffer, pH 7.5, for 10 min. The reaction was made visible by using a mixture of 3 mg 3,3'-diaminobenzidine (DAB; Sigma, St. Louis, MO, USA) in 15 mL 0.05 mol/L Tris-HCl buffer, pH 7.5, and 36 μL 1% hydrogen peroxide for 10-20 min, and rinsed in PBS, pH 7.4. The sections were dried overnight at room temperature and then mounted with entelan in xylene for light microscopy. They were not counterstained for better visualization of the DAB reaction product.

Sections incubated with non-immune sera instead of the primary antibodies were used as negative controls.

Determination of Microvessel Density and Mast Cell Density

Microvessel density (MVD) of highly vascularized tumor areas, so-called hot spots of tumor neovascularization, were evaluated as suggested by Lackner et al.[10] and by Tanigawa et al.[9] Using light microscopy, the 'hot spots' were localized in the invasive front of the colorectal cancer. The stained sections were screened first at low power (×100) to identify the areas of highest vascularization within the tumor invasive front (hot spots). Microvessel counts were performed at ×320 magnification in five fields of vision (corresponding to an investigated area of 0.74 mm2) with an ocular micrometer, and for each case the mean 'hot spot' MVD was noted. The systematic MVD was also evaluated on five consecutive fields of vision in the invasive front of the cancers and for each case the mean MVD was noted. Positively stained blood vessels with lumen as well as cell clusters without a lumen and single cells were considered as individual vessels.

In order to analyze the correlation between angiogenesis and mast cell response of the host, mast cells were also counted in five microscopic fields at a magnification ×320 within the areas of highest MVD at the invasive tumor front. The mast cell density (MCD) was evaluated also on five consecutive fields of vision in the invasive front of the cancers. The mean values of MCD for each case were noted.

Statistical Analysis

Statistical analyses were performed using StatView v.4.53 for Windows (Abacus Concepts, Berkley, CA, USA). The descriptive statistical tests, including the mean, standard deviation, and median, were calculated according to the standard methods. Kolmogorov-Smirnov test, Lilliefors test and Shapiro-Wilks' W-test were used for analyzing the normality of the distribution. The non-parametric Mann-Whitney U-test and Kruskal-Wallis test were used to evaluate the significance of the differences of the mean ranks. Non-parametric Spearman's rank correlation test was applied to study the correlation between the MCD and MVD. Cumulative survival curves were drawn by the Kaplan-Meier method and the difference between the curves analyzed by the Mantel-Cox (log-rank) test. The Cox's proportional hazard model of the StatView package was used in the multivariate analysis, performed with factors which were significant in the univariate analysis. Hazard ratios (HR) and their 95% confidence intervals (95% CI) were calculated. Factors with P < 0.05 were considered statistically significant.

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