Annals of Diagnostic Pathology
Volume 16, Issue 1 , Pages 1-6, January 2012

Association of mast cells with microvessel density in urothelial carcinomas of the urinary bladder

  • Aysegul Sari, MD

      Affiliations

    • Pathology Department, Izmir Ataturk Researh and Training Hospital, Izmır, Turkey
    • Corresponding Author InformationCorresponding author. Tel.: +905325265215.
    • ,
  • Aylin Calli, MD

      Affiliations

    • Pathology Department, Izmir Ataturk Researh and Training Hospital, Izmır, Turkey
    • ,
  • Fulya Cakalagaoglu, MD

      Affiliations

    • Pathology Department, Izmir Ataturk Researh and Training Hospital, Izmır, Turkey
    • ,
  • Aysegul Aksoy Altınboga, MD

      Affiliations

    • Pathology Department, Izmir Ataturk Researh and Training Hospital, Izmır, Turkey
    • ,
  • Kaan Bal, MD

      Affiliations

    • Department of 2nd Urology, Izmir Ataturk Researh and Training Hospital, Izmır, Turkey

published online 24 October 2011.

Article Outline

Abstract 

This study aims to investigate the relation of mast cell (MC) accumulation with tumor grade and stage in urothelial carcinomas of the urinary bladder and to determine its relationship with angiogenesis. A total of 78 urothelial carcinomas obtained by transurethral resection were investigated immunohistochemically by using c-Kit (CD117) and anti-CD34. The correlation between MC counts and microvessels was evaluated and compared with histopathologic parameters including tumor stage and grade. There were significant correlations between MC counts, grade, and stage (P < .05; r = 0.69 and 0.63, respectively). However, MC counts in adjacent nontumoral bladder mucosa significantly were higher than the MC counts in tumoral zone (P < .001). On the other hand, significant correlation was found between the number of MCs in tumoral zone and the microvessel density (P < .05, r = 0.56). The results of our study suggest that c-Kit positive MCs in tumoral zone may contribute to tumor angiogenesis and play a significant role in tumor growth and invasion. Further studies are needed to support these observations.

Keywords: Mast cell, c-Kit positive, Angiogenesis, Carcinoma, Urothelial, Bladder

 

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1. Introduction 

Tumor angiogenesis, the proliferation of new vessels from preexisting vessels around and within a tumor, is mandatory for tumor growth, invasion, and metastasis [1]. The impact of angiogenesis on tumor progression has been well established. Microvessel density (MVD), the commonly used histologic measure of angiogenesis, is found to be an independent predictor of survival in various types of tumors such as breast, prostate, lung, larynx, including bladder [2], [3], [4], [5], [6]; however, there are conflicting studies on the correlation between angiogenesis and prognosis in superficial urothelial carcinomas [7], [8], [9], [10], [11].

The effect of stroma and chronic inflammation surrounding the tumor in cancer development and progression has been reported frequently [12], [13]. Many cell types, such as host immune cells, are thought to play crucial role. Among these, mast cells (MCs) may promote tumor development through many different ways. Mast cells could contribute to tumor growth by facilitating angiogenesis through heparin-like molecules, growth factors (fibroblast growth factor 2, vascular endothelial growth factor [VEGF]), cytokines (tumor necrosis factor-, interleukin [IL]-8), and histamine [14], [15], [16], [17]. This is a major point linking MCs to cancer [18], [19]. Mast cells are also rich in metalloproteases that contribute to most of proteolytic components necessary for tumor invasiveness [14].

Mast cell infiltration and activation in tumors are reported to be mainly mediated by tumor-derived stem cell factor (SCF) and its receptor c-Kit on MCs [13], [18], [19]. Stem cell factor is the chief promoter of the c-Kit signaling pathway for the MC growth, differentiation, and maturation from bone marrow and peripheral blood progenitors and arranges the multiple functions of mature MCs [13], [20]. On animal models using tumor-bearing mice, it has been demonstrated that both anti-SCF and anti–c-Kit antibodies effectively attenuated the infiltration of bone marrow–derived MCs into the tumor, pointing out that MC migration is mainly mediated by the SCF/c-Kit axis. Stem cell factor/c-Kit signal stimulates mast cell–induced remodeling of tumor inflammatory microenvironment and activates MCs to trigger the immunosuppression. [13]. Stem cell factor/c-Kit signal can also induce MCs to produce matrix metalloproteinase 9 (MMP9) and a large number of proinflammatory factors such as IL-6, tumor necrosis factor α, VEGF, and Cyclooxygenase-2 (COX2). However, proinflammatory genes were found to be up-regulated in MCs only if SCF is present at high concentrations but not at low concentrations, and this up-regulation could be suppressed by anti–c-Kit antibody [13].

Mast cell count has been found to be associated with enhanced growth and invasion of variety of human cancers including colon, stomach, breast, oral cavity, lung, and laryngeal cancers [5], [15], [21], [22], [23], [24]. However, data are limited evaluating the role of MCs in urothelial carcinomas [25], [26], and furthermore, the relationship of MCs with angiogenesis has not been yet investigated in urothelial carcinomas.

Urothelial carcinoma of the urinary bladder is the fifth most common cancer in the general population and the second most common cancer of the genitourinary tract. Most patients (70%-80%) with newly diagnosed urothelial carcinoma of the bladder present with superficial papillary tumors, up to 70% of these patients experience recurrent disease, and approximately 15% progress to muscle-invasive carcinoma. Although there is an intensive search for novel molecular and biologic markers to predict the risk of recurrence or progression in urothelial carcinomas, pathologic stage and grade still remain to be the most valuable prognostic factors [10], [11].

In this study, we aimed to investigate the relation of c-Kit positive MC population with angiogenesis and to determine whether c-Kit–positive MC count has any relation with clinicopathologic parameters such as tumor grade and pathologic stage in urothelial carcinomas of the bladder.

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2. Materials and methods 

We examined a total of 78 urothelial carcinoma of the urinary bladder from the archives of the Pathology Department of Izmir Ataturk Training and Research Hospital 2007 and 2010. All tissues were from transurethral resections. In 31 specimens, fragments of nontumoral adjacent bladder mucosa were also present. None of the patients had received preoperative therapy. Hematoxylin-eosin–stained slides obtained from formalin-fixed, paraffin-embedded material were reevaluated. The tumors were classified according to the 2004 World Health Organization histologic classification of tumors of the urinary tract [27] and staged by the American Joint Committee on Cancer system of 2002 [28].

2.1. Immunohistochemical staining 

Formalin-fixed, paraffin-embedded tissue sections of 4 to 5 μm from each representative block were placed onto polylysine-coated slides for immunohistochemical staining with CD34 (monoclonal mouse antibody, dilution 1:40; Dako, Denmark) and CD117 (polyclonal, dilution 1:400; Dako). Immunohistochemistry was performed by using the standard streptavidin-biotin-peroxidase complex (Leica Microsytems, Buffalo Grove, IL) method using the Leica Bond-Max autoimmunostainer with Bond Polymer Refine Detection Kits and heat-induced epitope retrieval pH 8.0 (Bond max ER2 [EDTA] solution; Melbourne, Australia) for 15 minutes.

2.2. Determination of MC counts and MVD 

The stained sections were first scanned at low magnification (×40 and ×100) to identify the areas with the highest number of microvessels in the tumoral zone (hot spots). Three most vascularized areas were counted within a ×400 microscopic field, and the mean microvessel count was noted as the MVD. Any cell with brown-cytoplasmic staining was considered a single, countable microvessel (Fig. 1), and the lumen diameter, which was smaller than approximately 8 blood cells, was taken into account [11]. Mast cells were also counted with the same method both in the tumoral zone (Fig. 2, Fig. 3), in nontumoral adjacent bladder mucosa. The mean MVD and MC counts were recorded for each case.

2.3. Statistical analysis 

Continuous data were presented as median (minimum-maximum) values. Comparison between the 2 groups with continuous variables was performed by the Mann-Whitney U test, whereas the Kruskal-Wallis test was used for multiple groups (more than 2). Where the Kruskal-Wallis test produced significant results, the Mann-Whitney U test was used for pairwise group comparisons. Spearman correlation coefficients were calculated to quantify the association between variables. Differences between the categorical data were analyzed by the χ2 test. We performed Wilcoxon signed rank tests, which test the difference between paired measurements such as the comparisons of MCs in tumoral zone vs adjacent nontumoral bladder tissue. Two-tailed P values less than .05 were considered statistically significant. Calculations were performed by the SPSS statistical package (version 13.0; SPSS Inc, Chicago, Ill).

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3. Results 

There were 78 patients (72 men, 6 women). Median ages of the patients were 66 years (19-87 years). According to the tumor grade, 35 (45%) subjects were low-grade and 43 (55%) were high-grade tumors. According to the pathological stage (pT), of 78 tumors, 32 (41%) were pTa, 26 (33%) were pT1, and 20 (26%) were pT2 tumors. A significant amount of nonneoplastic bladder mucosa was present in 31 specimens adjacent to carcinomas.

The median MC counts in tumoral zone and adjacent nontumoral bladder tissue were 9 (1-28.3) and 14.65 (7.3-22.3), respectively. The median MVD was 15.95 (4.3-40). Table 1 demonstrates the demographical and histopathologic characteristics of the study group.

Table 1. Demographic and histopathologic characteristics of the study group
Age (y)a66 (19-87)
Sex (male/female)72/6
Tumor grade, n (%)
Low35 (45)
High43 (55)
Tumor stage, n (%)
pTa32 (41)
pT126 (33)
pT220 (26)
MC counts
Tumoral zone9 (1-28.3)
Adjacent nontumoral bladder tissue14.65 (7.3-22.3)
MVD15.95 (4.3-40)

aData were presented as median with minimum-maximum values.

3.1. Relation of MC count in tumoral tissue with other parameters 

There were significant correlations between c-Kit–positive MC counts in tumoral tissue, grade, stage, and MVD (P < .05; r = 0.69, 0.63, and 0.56, respectively).

3.2. Relation of MVD with other parameters 

Microvessel density was significantly correlated with grade, stage, and MC counts in tumoral tissue (P < .05; r = 0.61, 0.7, and 0.56, respectively).

3.3. The comparisons of MC counts and MVD according to the tumor stage and grade 

c-Kit–positive MC counts in tumoral zone were significantly different between stages (Kruskal-Wallis P < .001). The pairwise comparisons revealed that MC counts are significantly lower in pTa than pT1 and pT2 tumors. However, there were no differences between pT1 and pT2 regarding MC counts (median counts: pTa = 5.3 [1-18], pT1 = 10.8 [2.6-28.3], and pT2 = 15.3 [5-23.6]; P < .001, pTa vs pT1; P < .001, pTa vs pT2; and P = .12, pT1 vs pT2).

Microvessel counts were also significantly different between stages (Kruskal-Wallis P < .001).Microvessel density were significantly lower in pTa than pT1 and pT2 (median counts: pTa = 9.6 [5.3-22.6], pT1 = 17.8 [4.3-38], and pT2 = 26.4 [12-40]; P = .001, pTa vs pT1 and P < .001, pTa vs pT2). In addition, MVD in pT1 was significantly lower than pT2 (P < .001, pT1 vs pT2).

Both MC counts and MVD were significantly lower in low-grade tumors than high-grade tumors (P < .05). Table 2 shows the comparisons of MC counts and MVD according to the tumor stage and grade.

Table 2. The comparisons of MC counts and MVD according to the tumor stage and grade
pTapT1pT2P
MC count5.3 (1-18)10.8 (2.6-28.3)15.3 (5-23.6)<.001
MVD9.6 (5.3-22.6)17.8 (4.3-38)26.4 (12-40)<.001

Low gradeHigh gradeP
MC count5.3 (1-13)14 (5-28.3)<.001
MVD9.6 (5.3-21)22.6 (4.3-40)<.001

Data were presented as median with minimum-maximum values.

3.4. Comparisons of MC counts in tumoral tissue and adjacent nontumoral bladder tissue 

Comparison of c-Kit–positive MC count in tumoral tissue and adjacent nontumoral bladder tissue was significantly different (P < .001).

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4. Discussion 

In this study, we investigated the role of c-Kit–positive MC counts in urothelial carcinoma of the urinary bladder and its association with angiogenesis. We found that the MC counts in the tumoral zone were higher in high-grade and advanced stage tumors and that MC counts were significantly correlated with angiogenesis.

Angiogenesis is not only requisite for healing, reproduction, and embryonic development but also a needful process that plays an important role in tumor growth, progression, and metastases [1], [29]. Angiogenesis can be assessed by determining MVD of the tumor by counting the number of vessels within a vascular area from sections stained with specific antibodies for highlighting vessels [10]. Concerning bladder tumors, MVD has been found to be significantly correlated with poor prognosis in invasive bladder carcinoma [6], [30], [31], but data are conflicting in superficial bladder tumors [7], [8], [9], [10], [11]. This might be because of the mostly small and papillary nature of the disease [10] and/or different kinds of methods used for assessing microvessel counts [32]. In our study, MVD showed a statistically significant difference between grade and stage.

There is substantial evidence to reinforce the view that angiogenesis and inflammation are mutually dependent [19]. The critical role of inflammatory cells and, among these, MCs in regulating tumor progression and angiogenesis is well known [16]. Tumors in MC-deficient mice both had reduced angiogenesis and fewer metastases [33]. In spite of the evidence suggesting that MCs can promote tumorigenesis and tumor progression, there are some studies held on different tumors including breast, colon [17], [34], [35], as well as experimental tumor models in which MCs seem to have functions that favor the host [19]. This potential dual role of MCs could be caused by their ability to secrete either the individual granules or distinct mediators selectively [14]. Granulated MCs and their granules, but not degranulated MCs, were able to stimulate a potent angiogenic reaction in the chick embryo chorioallantoic membrane (CAM) assay [19]. The only way MCs could assist the tumor is if the secretion of beneficial molecules from MCs could be secreted selectively without degranulation.

Mast cells could participate in tumor rejection by producing molecules, such as IL-1, IL-4, IL-6, and tumor necrosis factor α, which kill tumor cells [14]. The state of mast cell differentiation, activation, and also local stromal conditions are also important regarding the type of molecules it could synthesize and store, which might explain the dual role of MCs [14], [17]. Furthermore, in spite of common features, MCs also show marked differences in their phenotypic expression in different species and anatomical sites, a phenomenon called MC heterogeneity [36].

Although MCs could be recruited by various chemoattractants, MC accumulation and activation in tumors are demonstrated to be mainly mediated by tumor-derived SCF and its receptor c-Kit on MCs [13], [19]. In hepatocarcinoma model, Huang et al [13] were able to demonstrate that MCs failed to migrate into SCF knockdown tumor and that anti–c-Kit antibodies impaired the infiltration of MCs into the tumor. Therefore, the SCF/c-Kit pathway seems to be a very important target for tumor therapy [13].

The evaluation of MCs in bladder carcinoma is scant with only 2 studies presented to date [25], [26]. Gupta [25] was the first to mention MC in urinary bladder. They found that average number of MCs decreased with an increase in grade in urothelial carcinomas [25]. Serel et al [26] found that MCs accumulated in lamina propria of the superficial tumors and no correlation was demonstrated between grade and MC counts. However, the association of MC counts and stage was not mentioned in any of these studies. In both of the studies, toluidine blue was used to highlight the MCs.

In our study, we found a statistically significant difference between grade and stage of urothelial carcinomas in regard to c-Kit–positive MC counts. We also found a significant correlation between MC counts and MVD supporting the suggestion that MCs have a role in tumor-associated angiogenesis. Mast cells were significantly higher in noninvasive tumors (pTa) than invasive tumors (pT1 and pT2). However, among invasive tumors, both MC counts did not show statistically significant difference between tumors with lamina propria invasion (pT1) and muscularis propria invasion (pT2). This could be because MC infiltration into a developing tumor induces an “angiogenic switch” at early stages in tumorigenesis and at later stages tumor cells take the control of growth and angiogenesis and the growth becomes MC independent, with still containing significant amount of MCs [37]. We also observed that, in adjacent nontumoral bladder tissue, the MC counts were higher than the MCs of the tumoral zone. The increased number of MCs in the adjacent nontumoral mucosa might take part in inhibiting the primary tumor [26]. This could be because of the state of MC differentiation, activation, and as well as local stromal conditions, which can alter the phenotypic behavior and secretory processes of MCs [14]. Furthermore, proinflammatory factors in MCs were shown to be up-regulated only in the presence of high concentration of SCF [13], so irrelevant with the number of MCs, the probable low concentration of SCF in nontumoral adjacent stroma could restrain MCs to secrete molecules for tumor growth. Regarding the association of MC counts with grade in urothelial carcinomas, discrepancies could be caused by the different methods of counting and different staining technologies for labeling the MCs and that toluidine blue might not demonstrate all the MCs [38].

In conclusion, we found that MC infiltration in tumoral zone was correlated with tumor grade, stage, and MVD in urothelial carcinomas of the bladder. The results of our study suggest that MCs contribute to tumor angiogenesis and play a significant role in tumor growth and invasion. However, the findings of our study should be supported with further studies regarding the complex role of MCs in neoplasia and the limited data with controversial findings on bladder cancer. Therefore, MCs, to be a possible target for controlling tumor growth in bladder carcinoma, need further investigation.

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PII: S1092-9134(11)00104-3

doi:10.1016/j.anndiagpath.2011.07.001

Annals of Diagnostic Pathology
Volume 16, Issue 1 , Pages 1-6, January 2012

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