|Year : 2013 | Volume
| Issue : 4 | Page : 701-705
Angiogenesis and mast cell density as predictors of patient survival in squamous cell carcinoma of lung
Ehsan Ullah1, Abdul Hannan Nagi2, Muhammad Ashraf3
1 Department of Pathology, Quaid-e-Azam Medical College Bahawalpur, Pakistan
2 Pathology, University of Health Sciences Lahore, Pakistan
3 Histopathology, Gulab Devi Chest Hospital Lahore, Pakistan
|Date of Web Publication||11-Feb-2014|
Department of Pathology, Quaid-e-Azam Medical College Bahawalpur - 63100
Source of Support: University of Health Sciences Lahore, Pakistan, Conflict of Interest: None
Background: Measuring the microvascular and mast cell density in squamous cell carcinoma of lung and correlating them with the patient survival may be helpful to guide the use of cancer chemotherapeutic agents which target molecular mechanisms of tumour angiogenesis and mast cells.
Materials and Methods: It was an observational study. It included 39 newly diagnosed, adult patients of pulmonary squamous cell carcinoma. Angiogenesis was determined by Chalkley's method after immunohistochemical staining of micro-vessels with CD34. Mast cells per HPF were counted in Tolouidine blue stained sections.
Results: Mean age of the patients was 58.33 ± 9.14 years. Male to female ratio was 9:1. Most (92.3%) patients were current smokers. Majority of tumours (71.8%) were localised to major bronchi and/or near to hilum and many of them (74.4%) were poorly differentiated. Mean micro-vascular density was 11.80 ± 3.66 per HPF which showed strong negative correlation (r = -0.481, p =0.002) between microvascular density (MVD) and tumour grade. Mean mast cell density was 1.60 ± 2.04 which showed strong negative correlation (r=-.683, p =0.0001) with grade. Angiogenesis and mast cell density were found to be positively correlated (r=0.439, p =0.005). High MVD, but not the MCD was associated with poor survival.
Conclusion: Angiogenesis and mast cell density are positively correlated with each other however; only high MVD is associated with decreased survival. Thus, the anti-angiogenic agents may be useful in squamous cell carcinoma lung, especially the well differentiated tumours.
Keywords: Angiogenesis, lung cancer, mast cell, squamous cell carcinoma
|How to cite this article:|
Ullah E, Nagi AH, Ashraf M. Angiogenesis and mast cell density as predictors of patient survival in squamous cell carcinoma of lung. J Can Res Ther 2013;9:701-5
|How to cite this URL:|
Ullah E, Nagi AH, Ashraf M. Angiogenesis and mast cell density as predictors of patient survival in squamous cell carcinoma of lung. J Can Res Ther [serial online] 2013 [cited 2020 Jan 25];9:701-5. Available from: http://www.cancerjournal.net/text.asp?2013/9/4/701/126487
| > Introduction|| |
Despite extensive work with extremely sophisticated techniques available today and untiring intellectual efforts on preventive and therapeutic aspects, Lung Cancer (LC) remains the most fatal cancer worldwide.  LC is a group of numerous histologically distinct entities however; squamous cell carcinoma (SqCC) is the commonest morphological type across the globe as well as Pakistan. , Along with small cell carcinoma, it is the most strongly correlated with smoking. ,,
Neovascularization or angiogenesis is critical for the development, growth and progression of solid tumors, including SqCC of lung. ,, Thus, angiogenic pathways have evolved as an important biologic target to hit the tumour proliferation. About 40 years of research have led from the discovery of a factor capable of inducing tumour angiogenesis to the concept of targeted anti-angiogenesis therapy. ,,, Several pathways for tumour angiogenesis have been recognized, including the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) pathways.  However, the tumour infiltrating mast cells (MCs) have exhibited their complex role in mediating angiogenesis as well as tumour growth and proliferation. Mast cells have been observed to be pro-angiogenic through release of VEGF, FGF-β and other chemical mediators. , In addition, mast cells modulate immune responses by dampening immune rejection or directing immune cell recruitment, depending on local stimuli. Studies suggest that MCs may serve as a novel therapeutic target for cancer treatment and that inhibiting MC function may lead to tumor regression. 
This study aims at describing the relationship between angiogenesis, mast cells and patient survival in SqCC of lung.
| > Materials and Methods|| |
It was an observational, descriptive study. Tumour samples were obtained from the patients who underwent biopsy procedures or surgical removal of their lung tumours diagnosed as squamous cell carcinoma according to WHO classification (1999) during a period from March 2010 to August 2011. The study was conducted after taking approval from Ethics Committee of the University. These patients had not been treated with neoadjuvant chemotherapy or irradiation therapy before tumour tissues were obtained. However, all patients were offered chemotherapy and palliation since most of the patients were harbouring advance stage disease at the time of presentation. Paraffin blocks containing sufficient formalin-fixed tumour for sampling were available from 39 tumours.
Microvascular density (MVD) or angiogenesis per section was measured using immunohistochemical staining with a CD34 monoclonal antibody (Clone QBEnd10; BioSB, USA), which required phosphate buffer saline based antigen retrieval and 30 min incubation of the primary antibody (prediluted) at 37°C. MVD of each case of squamous cell carcinoma was defined as the mean number of CD34 + vessels per section. The slides were mounted and examined using a bright-field microscope. Since the sections were small, we did not identify a hot spot to count the CD34 + vessels, but counted vessels in the whole section. A positive reaction was indicated by a reddish-brown precipitate in the cytoplasm. For quantification of tumour MVD, highly vascular areas were initially identified by scanning tumour sections using light microscopy at low power. Vessels count was assessed in areas of the tumour containing the highest numbers of capillaries and small venules, based on the criteria of Weidner et al.  Vessels in five high-power fields (400 × magnifications) were counted by two independent investigators without knowledge of the patient outcome. An average value of the two scores was presented in the current study. Mast cell counts per high power field (HPF) were calculated in the same way, in Toulodine blue stained sections.
All the data was collected on specially designed proforma. The data was analysed with the help of SPSS version 17. Mean ± S.E. of mean was given along with 95% confidence intervals for quantitative variables. Frequencies and percentages were given for qualitative variables. Means were compared with the help of One-way ANOVA and intra-group variation was sought by applying Post-hoc Tukey HSD test. Correlation between continuous variables was determined with Pearson's correlation whereas; Spearman's rho was used to measure the correlation co-efficient when one or more of the variables were rank-scale. Kaplan-Meier survival curves were plotted and Log Rank (Mantel-Cox) tests were applied to observe the effect of angiogenesis, mast cell density and histological grade of the tumour on survival of the patients. p <0.05 was considered to be statistically significant.
| > Results|| |
Majority of the patients (n=35, 89.3%) were men. Only four patients (10.3%) were females. Mean age of the patients was 58.33 ± 9.14 years. History of current or past smoking of more than 10 pack years was positive in most (92.3%) patients. On histological examination, four cases (10.3%) were labelled as well-differentiated, eight cases (20.7%) as moderately-differentiated and remaining 27 cases (70.3%) as poorly-differentiated tumours. Most of the patients (n=33, 82.5%) presented at advance stage (TNM-III or IV). Only five cases (15.0%) presented at early stage disease (TNM-I or II), however due to the central location of tumour and based local treatment protocols, all patients underwent chemotherapy and palliative care. Median survival was 8 m (range: 1 - 15 m). Patient characteristics are given in [Table 1].
Mean microvascular density or angiogenesis was 11.80 ± 3.66 microvessels per high power field as shown in [Figure 1]. We divided the tumours into two categories viz. having high or low microvascular where the dividing cut off was the mean of the variable. High microvascular density was noticed in more commonly in well-differentiated (75.0%) and moderately differentiated (75.0%) tumours as compared to poorly differentiated squamous cell carcinoma (18.52%). This association was statistically significant (Fischer's Exact test, p=0.002). There was strong negative correlation between microvascular density and histological grade of the tumour was also observed (=-0.554, p=0.001). Significantly reduced survival was observed among patients with high MVD (p=0.001) as shown in [Figure 2].
|Figure 2: Kaplan-Meier Survival Curve for High vs. Low Microvascular Density in Squamous Cell Carcinoma of Lung (p =0.001)|
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Mean mast cell density was 1.60 ± 2.04 mast cells per high power field as shown in [Figure 3]. As noticed for MVD earlier, the MCD was found to be significantly higher in well-differentiated and moderately-differentiated tumours than poorly-differentiated ones (Fischer's Exact test, p=0.002) thus strong negative correlation between MCD and histological grade of SqCC was found (r=-0.683, p=0.0001). High MCD was also found to be associated with early stage (TNM IandII) as compared to late stage (TNM IIIandIV) as shown in [Table 2]. However, no significant effect of MCD on patient survival was observed (p=0.268) as shown in [Figure 4].
|Figure 3: Mast cells in Squamous Cell Carcinoma of Lung (Toulodine blue)|
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|Figure 4: Kaplan-Meier Survival Curve for High vs. Low Mast Cell Density in Squamous Cell Carcinoma of Lung (p =0.268)|
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|Table 2: Relationship between angiogenesis, mast cell density and clinicopathological parameters|
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Mean MVD in the tumour specimens having high MCD was 12.50 ± 2.86 /HPF as compared to 8.96 ± 2.32 /HPF the tumour specimens having low MCD thus, a statistically significant positive correlation (r=0.439, p=0.005) was found between the two which, in other words it means that mast cells have pro-angiogenic role in SqCC of lung.
| > Discussion|| |
Authors have presented clinicopathological features of squamous cell carcinoma of lung along with the correlations between angiogenesis, mast cell density and histological grades of this tumour. In current study, the average age of patients at the time of presentation was 58.33 (1.69) years, which was similar to the findings reported in other studies conducted on lung cancer patients, from within the country. , Majority of the patients were males i.e. male to female ratio was 9:1 in current study. Another study from Punjab province reported a male to female ratio of 7.6:1 for lung cancer where more than half (53%) of the cases were histologically diagnosed as squamous cell carcinoma.  While a study from India, a neighbor country also showed similar male to female ratio of 5.7 - 11.3:1. , Most of the patients (92.30%) were smokers with average duration of smoking more than 10 years. It is universally accepted and established fact that squamous cell carcinoma is one the two most strongly smoking-associated types of lung cancer - the other being small cell carcinoma. 
Histologically, majority of the tumours (70.3%) in current study were found to be high grade (poorly differentiated). According to Naseem et al. 74% cases of squamous cell carcinoma of lung were found to be poorly differentiated tumours.  Most of the cases presented at advance stage where surgical intervention could not be offered, a finding consistent with most of other investigators. ,
Angiogenesis was quantified in terms of microvascular density and described as microvessels per high power field. In current study, a mean of 11.80 ± 3.66 microvessels were seen per high power field of intra-tumoural zones of squamous cell carcinoma of lung. It was also observed that microvascular density was highest among the tumours showing excellent differentiation and least among the poorly differentiated tumours. High MVD was associated with significantly decreased patient survival (p=0.001) as by Kaplan-Meier survival curve shown in [Figure 3]. A recent study found an average MVD of 10.18 ± 4.70 in the intra-tumoural zones of oral squamous cell carcinoma however no significant association of MVD with tumour grade was observed.  Studies have shown that angiogenesis, as quantitation according to MVD, plays significant clinicopathologic roles in cancer patients. Possibly, MVD can be an independent, highly significant and accurate prognostic indicator in cancer patients. , Another study has shown microvascular density as independent prognostic factor of non-small cell carcinoma of lung along with size of primary tumour.  A recent study reported that high MVD was associated with poor survival in squamous cell carcinoma of larynx. 
Mast cells have an established role in tumour kinetics and angiogenesis.  It has been observed that mast cells enhance angiogenesis in various cancers. , It is advocated that some of the mast cell molecules can be targeted with the pharmaceutical agents that may modulate their function and thus prove useful in the treatment of cancer.  Some of the important anti-cancer drugs which exert their anti-tumour effects by modulating mast cell molecules include Imatinib which is hypothesized to virtually ablate tumour mast cells by through c-KIT receptor modulation. , In current study, average mast cell density in the intra-tumoural zones of squamous cell carcinoma of lung was found to be 1.60 ± 2.04 mast cells per high power field. It was also found that MCD was positively correlated with MVD (r=0.439, p=0.005) thus suggesting mast cells' pro-angiogenic role. Tomita and coworkers also observed similar interdependence between mast cells and angiogenesis.  Some recent studies further advocate's angiogenesis potentiating characteristics of mast cells. , In current study MCD was found to have negative correlation with histological grade of SCC of lung. However, there was no significant effect of MCD was observed on patient survival (Mantel cox = 0.268) as shown in [Figure 4]. Similar finding has been reported by Niczyporuk and coworkers.  However, studying various sub-types of mast cells in intra-tumoural and peri-tumoural zones of non-small cell carcinoma of lung, some investigators found low MCD to be associated with worse prognosis, thus adding complexity to our understanding about exact role of tumour mast cells in angiogenesis and tumour progression.  As indicated by other investigators, authors also suggest that mast cell sub-types should be studied along with mast cell molecules like VEGF, microvessel density and patient survival in SqCC of lung and other human cancers since evidence from experimental studies proves that mast cells serve as novel therapeutic target for cancer treatment and the controversial results may stem from different methods adopted by various investigators. ,,
| > Conclusions|| |
Current study showed that angiogenesis and mast cell density are positively correlated with each other however; only high MVD is significantly associated with decreased survival of patients. Thus, it is concluded that anti-angiogenic agents may be useful in squamous cell carcinoma lung, especially the well differentiated tumours.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]