|Year : 2012 | Volume
| Issue : 4 | Page : 537-541
Angiogenesis and mast cell density in invasive pulmonary adenocarcinoma
Ehsan Ullah, Abdul H Nagi, Raees A Lail
Department of Morbid Anatomy and Histopathology, University of Health Sciences, Khayaban-e-Jamia, Lahore, Pakistan
|Date of Web Publication||29-Jan-2013|
Department of Pathology, Quaid-e-Azam Medical College, Bahawalpur - 63100
Source of Support: University of Health Sciences Lahore, Pakistan,, Conflict of Interest: None
Introduction: Angiogenesis and mast cells affect the behavior of pulmonary adenocarcinoma. Measuring the angiogenesis and mast cell density (MCD) and their effect on survival of the patients may be helpful to guide the use of cancer chemotherapeutic agents which target tumor angiogenesis and mast cells.
Materials and Methods: It was a descriptive study, conducted at Gulab Devi Chest Hospital and University of Health Sciences Lahore. It included 23 newly diagnosed, adult patients of invasive pulmonary adenocarcinoma. Clinical history was obtained and biopsy specimen was processed. Angiogenesis was determined by immunohistochemical staining with CD34. Mast cells were counted in toluidine blue stained sections. Patients were followed-up till death.
Results: Mean age of the patients was 54.83 ± 2.799 years. Majority (60.9%) were males. Only 39.1% patients were smokers. Morphologically, large proportions of tumors (73.9%) were nonmucinous and moderately or poorly differentiated. Majority (69.6%) of patients presented at advanced stage (Tumor-Node-Metastasis staging (TNM )III and IV). Mean microvascular density (MVD) was 13.04 ± 1.12 and mean MCD was 3.26 ± 0.36 per high power field (HPF). High MVD was associated with poor differentiation and advanced stage and correlated with poor survival (P = 0.0001). High MCD was associated with high grade but not with the advance stage of disease. However, high MCD correlated with poor survival (P = 0.047). Moreover, MCD was positively correlated with angiogenesis (r = 0.727, P = 0.0001). Treatment did not affect the survival significantly (P = 0.069).
Conclusion: High angiogenesis and MCD predict poor survival and are positively correlated with each other and with the histological grades in pulmonary adenocarcinoma. High angiogenesis is also associated with advance TNM stage of disease.
Keywords: Lung cancer, adenocarcinoma, mast cell density, angiogenesis, patient survival
|How to cite this article:|
Ullah E, Nagi AH, Lail RA. Angiogenesis and mast cell density in invasive pulmonary adenocarcinoma. J Can Res Ther 2012;8:537-41
| > Introduction|| |
Angiogenesis, that is, formation of new blood vessels, plays a fundamental role in tumor growth.  Folkman hypothesized that tumor cannot grow beyond a diameter of 2 mm without neoangiogenesis.  In fact, a number of antiangiogenic medications are being used as chemotherapeutic agents in many cancers including lung carcinoma. ,, Tumor angiogenesis is different and more complicated process than the new blood vessel formation in nontumor conditions. It is tightly regulated by the tantalizing balance between several molecules, which enhance it, and a number of other molecules, which inhibit it. These angiogenic mediators are released either by tumor cells themselves or by a variety of cells present in or homing into the host tissue. One of these cells is the mast cell. 
Measuring the angiogenesis and mast cell density (MCD) in pulmonary adenocarcinoma and correlating them with pathological features may be helpful to guide the use of antiangiogenic and mast cell modulating cancer chemotherapeutic agents.
| > Methods|| |
It was an observational, descriptive study. Tumor samples were obtained from consenting patients undergoing biopsy procedure or surgical removal of a lung tumor diagnosed as adenocarcinoma according to WHO classification (1999) from March 2010 to August 2011 in Gulab Devi Chest Hospital Lahore. These patients had not been treated with neoadjuvant chemotherapy or irradiation therapy before tumor tissues were obtained. However, 20 patients were offered chemotherapy and surgical excision was carried out in three cases in which tumors were in early stage and resectable. Paraffin blocks containing sufficient formalin-fixed tumor for sampling were available from 23 tumors. In addition, 27 cases of lung tissue samples, obtained from autopsy nonneoplastic specimens, were used as controls. Patient details are summarized for each tumor type in [Table 1].
Microvascular density (MVD) or angiogenesis per section was measured using immunostaining with a CD34-monoclonal antibody (Clone QBEnd10; BioSB, USA), shown in [Figure 1]a 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 adenocarcinoma 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 entire section. A positive reaction was indicated by a reddish-brown precipitate in the cytoplasm. For quantification of tumor MVD, highly vascular areas were initially identified by scanning tumor sections using light microscopy at low power. Vessels count was assessed in areas of the tumor containing the highest numbers of capillaries and small venules, based on the criteria of Weidner et al. Vessels in five high-power fields (200× 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 toluidine blue stained sections as shown in [Figure 1]c.
| > Statistical Analysis|| |
All the data were collected on specially designed proforma. The data were analyzed with the help of SPSS version 19 (IBM Inc.). University of Health Sciences Lahore, Pakistan. 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 ANOVA and intra-group variation was sought by applying post-hoc Tukey honestly significant difference (HSD) test. Correlation between continuous variables was determined with Pearson's correlation and Spearman's rho was used to measure the correlation coefficient when one of the variables was rank-scale. Kaplan-Meier survival curves were plotted and Log Rank (Mantel-Cox) tests were applied observe the effect of angiogenesis and MCD on survival. P value < 0.05 was considered to be statistically significant.
| > Results|| |
There were 14 male (60.9%) and 9 female (39.1%) patients, with a median age of 55 years (range: 25-80 years). With regards to histomorpholocal type, 17 cases (73.9%) were nonmucinous and 6 cases (26.1%) were mucinous pulmonary adenocarcinoma. Out of 17 nonmucinous tumors, 4 showed features of papillary adenocarcinoma, and 1 revealed signet-ring morphology. Overall, 6 (26.1%) tumors were well-differentiated, 10 (43.5%) were moderately differentiated, and 7 (30.4%) were poorly differentiated. Majority (69.6%) of tumors were late stage (TNM stage III+IV) and remaining (30.4%) were early stage (TNM stage I+II) according to Union of International Cancer Control (UICC) staging classification.  Most of the patients (n=20, 86.95%) were offered chemotherapy since they presented very late or the site of the tumor rendered them unresectable. Only three patients (13.05%) were offered surgical excision followed by chemotherapy.
Relationship between angiogenesis, mast cell density, and pathological findings
Mean MVD was 13.04 ± 1.12 (95% CI: 10.71-15.37) microvessels per HPF. We divided the tumors in two groups based on either low or high microvessel density, according to the criteria mentioned earlier. MVD was found to have significant association with histological grade (P = 0.001) and with TNM stage (P = 0.005) of the tumors as shown in [Table 2]. Mean MCD was 3.26 ± 0.36 (95% CI: 2.51-4.01) mast cells per HPF. MCD was significantly associated with histological grade of the tumor only (P = 0.021).
|Table 2: Relationship between angiogenesis, mast cell density, and clinicopathological parameters|
Click here to view
Angiogenesis, mast cell density, treatment, and outcome of patients
After 9 months of the follow-up, all 23 patients were reported dead. Average survival was 5.20 ± 0.46 months (range: 1-9 months). Effect of angiogenesis and MVD on survival of the patients was determined with the help of Kaplan-Meier curves. Mean survival in patients having high angiogenesis (3.45 ± 0.343 months) was significantly less than the mean survival in patients having low angiogenesis (6.79 ± 0.409 months) with a P value of 0.0001. Similarly, among the patients having high MCD, mean survival (3.81 ± 0.82 months) was significantly less than the mean survival in patients having low MCD (5.93 ± 0.46 months) with a P value of 0.047. Thus, high MVD as shown in [Figure 1]b and MCD as shown in [Figure 1]d were found to predict poor outcome.
Angiogenesis and MCD were also found to be positively correlated (r = 0.727, P = 0.0001) with each other showing that tumor mast cells may have proangiogenic role. Mean survival in patients receiving chemotherapy was 4.88 ± 0.45 months, which was apparently less than the mean survival in patients undergoing surgery (7.33 ± 1.42 months) followed by chemotherapy. However, the difference was insignificant (P = 0.069) in statistical terms.
| > Discussion|| |
In current study, clinicopathological findings of pulmonary adenocarcinoma were presented along with correlations between angiogenesis, MCD and tumor grades. Mean age of the patients at the time of diagnosis was 59.11 ± 3.64 years. A study from Shaukat Khanum Memorial Cancer Hospital Lahore reported the mean age at presentation to be 60.1 years for male and 57.5 years for female patients of lung cancer.  Naseem et al. showed that most of the patients included in their study (82/142) were of the age group 41-60 years.  Male to female ratio in current study population was 1.8:1. In western part of the world, male to female ratio for this disease is nearly 1:1 that is linked with the almost equally frequent tobacco smoking in men and women.  However, in Pakistan, various histological types of lung cancer are predominantly affecting males. , Only 37.5% patients gave history of smoking. Previous studies have also documented that adenocarcinoma is weakly associated with the history of smoking. , The most common presenting symptoms were cough, dyspnoea, and chest pain. Other frequent complaints included fever, hemoptysis, weight loss, and anorexia. This finding is consistent with many of the previous reports. [14-16]
Microscopically, solid (50%) mucinous (25%), papillary (16.7%), and signet ring cell (4.2%) variants of pulmonary adenocarcinoma were found. It is being advocated that accurate sub-classification of adenocarcinoma of lung is predictive of its biological behavior.  According to histological grading, it was divided into grade-1 (26.1%), grade-2 (43.5%). and grade-3 (30.4%) tumors. Previous studies in the country reported the frequency of adenocarcinoma to be 14.9% and 12.4%, respectively. , But both these studies reported a larger number of cases as nonsmall cell carcinoma undifferentiated type that may be masked by the actual high frequency of pulmonary adenocarcinoma. In current study, it was noticed that larger proportion of females were affected by adenocarcinoma than with any other histological type of lung cancer. This finding is consistent with other investigators. ,,
Numerous evidence 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. Although more data have been published about the prognostic significance of MVD as an adverse prognosis factor in nonsmall cell type of lung cancer (NSCLC), the entity which includes adenocarcinoma as well, the exact prognostic value of MVD remains unclear.  In a recent study by Zhao and colleagues, the survival of patients having high MVD was found to be significantly less than the patients having low MVD (P = 0.021). High MVD was the only significant independent prognostic factor of overall survival (P = 0.027) among the patients characteristics.  Angiogenesis was quantified in terms of MVD. Mean MVD was 13.04 ± 1.12 microvessels per HPF. MVD was significantly associated with tumor grade (P = 0.001) and TNM stage (0.005) of pulmonary adenocarcinoma. Angiogenesis is universally accepted as indicator of poor prognosis.  Antiangiogenic agents, for example, Bevacizumab are approved for advance stage nonsmall cell types of lung cancer and a number of phase-II clinical trials are underway for small cell carcinoma of lung. 
Mean MCD was 3.26 ± 0.36 mast cells per HPF which was found to be significantly different (P = 0.009) in various grades of adenocarcinoma. Intra-group variations between grade-1 and grade-2 and between grade-2 and grade-3 were also significant (P = 0.008 for both. MCD was found to have strong positive correlation (r = 0.586, P = 0.003) with histological grade of adenocarcinoma. Angiogenesis and MCD were also found to be positively correlated (r = 0.727, P = 0.0001) with each other. Eady et al. found a direct correlation between MCD and MVD since long.  However, mast cells have been neglected in cancer research from the late 19 th to the start of the 21 st century. , Now it is an established fact that mast cells strongly affect tumor kinetics and angiogenesis.  Moreover, it is advocated that many of the mast cell membrane receptors or molecules can be targeted with suitable pharmaceutical agents that may modulate their function and thus prove to be useful in the treatment of cancer.  Some of the important anticancer drugs which exert their antitumor effects by modulating mast cell molecules include Imatinib, Sorafenib, Suntinib, Pazopanib, Axitinib, Dasatinib, Nilotinib, Enzastaurin, Tanespimycin, etc. Most of these drugs affect c-KIT or other receptors and thus modulate mast cell functions. However, it is hypothesized that c-KIT inhibitor drugs can virtually ablate tumor mast cells. , Other pharmacological agents listed above have different mechanisms of action, but the one major effect of most of these agents is to reduce the mast cell counts. 
It is widely reported that multimodality treatment such as chemotherapy and irradiation combined with surgical resection improves the outcome in adenocarcinoma and other types of nonsmall cell lung cancer, as compared with surgery or chemotherapy alone. ,, However, majority of cases in the current study were unresectable at the time of diagnosis thus we are unable to measure the effect of various therapeutic modalities on the survival of patients.
| > Conclusions|| |
High angiogenesis and MCD predict poor survival and are positively correlated with each other and with the histological grades in pulmonary adenocarcinoma. High angiogenesis is also associated with advance TNM stage of disease. Thus, antiangiogengic agents and mast cell modulating chemotherapeutic medications may be useful in all cases of invasive pulmonary adenocarcinoma especially those having high histological grade.
| > References|| |
|1.||Sun S, Schiller JH. Angiogenesis inhibitors in the treatment of lung cancer. Crit Rev Oncol Hematol 2007;62:93-104. |
|2.||Folkman J. Tumor angiogenesis: Therapeutic implications. N Engl J Med 1971;285:1182-6. |
|3.||Abrams TJ, Lee LB, Murray LJ, Pryer NK, Cherrington JM. SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer. Mol Cancer Ther 2003;2:471-8. |
|4.||Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350:2335-42. |
|5.||Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006;355:2542-50. |
|6.||Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis - correlation in invasive breast carcinoma. N Engl J Med 1991;324:1-8. |
|7.||Groome PA, Bolejack V, Crowley JJ, Kennedy C, Krasnik M, Sobin LH, et al. The IASLC lung cancer staging project: Validation of the proposals for revision of the T, N, and M descriptors and consequent stage groupings in the forthcoming (seventh) edition of the TNM classification of malignant tumours. J Thorac Oncol 2007;2:694-705. |
|8.||Badar F, Meerza F, Khokhar RA, Ali FA, Irfan N, Kamran S, et al. Characteristics of lung cancer patients-the Shaukat Khanum memorial experience. Asian Pac J Cancer Prev 2006;7:245-8. |
|9.||Naseem N, Sadiq S, Nagi AH, Ashraf M. A pattern of carcinoma of lung as seen in a tertiary care hospital. Biomedica 2008;24:118-23. |
|10.||Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277-300. |
|11.||Iqbal ZH. Presentation of lung cancer in south of Punjab. Biomedica 1997;13:6-10. |
|12.||Khuder SA. Effect of cigarette smoking on major histological types of lung cancer: A meta-analysis. Lung Cancer 2001;31:139-48. |
|13.||Park SK, Cho LY, Yang JJ, Park B, Chang SH, Lee KS, et al. Lung cancer risk and cigarette smoking, lung tuberculosis according to histologic type and gender in a population based case-control study. Lung Cancer 2010;68:20-6. |
|14.||Rana F, Rana H, Gill J, Saeed K. Epidemiology of lung cancer in Pakistani patients - Our experience at Shaukat Khanum Memorial Cancer Hospital and Research Centre. Ann King Edward Med Uni 1998;4:47-9. |
|15.||Molassiotis A, Lowe M, Blackhall F, Lorigan P. A qualitative exploration of a respiratory distress symptom cluster in lung cancer: Cough, breathlessness and fatigue. Lung Cancer 2011;71:94-102. |
|16.||Cheville AL, Novotny PJ, Sloan JA, Basford JR, Wampfler JA, Garces YI, et al. Fatigue, dyspnea, and cough comprise a persistent symptom cluster up to five years after diagnosis with lung cancer. J Pain Symptom Manage 2011;42:202-12. |
|17.||Daniels M, Wright G, Russell P, Williams R, Conron M, Wainer M. Comprehensive adenocarcinoma sub-typing predicts outcome after lung cancer resection. Heart Lung Circ 2011;20:250-84. |
|18.||Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred EN, Moore DH, et al. Tumor angiogenesis: A new significant and independent prognostic indicator in early-stage breast carcinoma. J Natl Cancer Inst 1992;84:1875-87. |
|19.||Inoshima N, Nakanishi Y, Minami T, Izumi M, Takayama K, Yoshino I, et al. The influence of dendritic cell infiltration and vascular endothelial growth factor expression on the prognosis of non-small cell lung cancer. Clin Cancer Res 2002;8:3480-6. |
|20.||Zhao M, Gao FH, Wang JY, Liu F, Yuan HH, Zhang WY, et al. JAK2/STAT3 signaling pathway activation mediates tumor angiogenesis by upregulation of VEGF and bFGF in non-small-cell lung cancer. Lung Cancer 2011;73:366-74. |
|21.||Liam CK, Pang YK, Leow CH, Poosparajah S, Menon A. Changes in the distribution of lung cancer cell types and patient demography in a developing multiracial Asian country: Experience of a university teaching hospital. Lung Cancer 2006;53:23-30. |
|22.||Bremnes RM, Camps C, Sirera R. Angiogenesis in non-small cell lung cancer: The prognostic impact of neoangiogenesis and the cytokines VEGF and bFGF in tumours and blood. Lung Cancer 2006;51:143-58. |
|23.||Eady RA, Cowen T, Marshall TF, Plummer V, Greaves MW. Mast cell population density, blood vessel density and histamine content in normal human skin. Br J Dermatol 1979;100:623-33. |
|24.||Westphal E. Uber mastzellen. In: Ehrlich P, editor. Ferbenanlytische Untersuchungen Zur Histologie Und Klinik Des Blutes. Berlin: Hirschwald; 1891. p. 17. |
|25.||Murdoch C, Muthana M, Coffelt SB Lewis CE. The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev Cancer 2008;8:618-31. |
|26.||Galinsky DS, Nechushtan H. Mast cells and cancer - no longer just basic science. Crit Rev Oncol Hematol 2008;68:115-30. |
|27.||Nechushtan H. The complexity of the complicity of mast cells in cancer. Int J Biochem Cell Biol 2010;42:551-4. |
|28.||Caglar HB, Baldini EH, Othus M, Rabin MS, Bueno R, Sugarbaker DJ, et al. Outcomes of patients with stage III nonsmall cell lung cancer treated with chemotherapy and radiation with and without surgery. Cancer 2009;115:4156-66. |
|29.||Albain KS, Swann RS, Rusch VW, Turrisi AT 3rd, Shepherd FA, Smith C, et al. Radiotherapy plus chemotherapy with or without surgical resection for stage III non-small-cell lung cancer: A phase III randomised controlled trial. Lancet 2009;374:379-86. |
|30.||Butts CA, Ding K, Seymour L, Twumasi-Ankrah P, Graham B, Gandara D, et al. Randomized phase III trial of vinorelbine plus cisplatin compared with observation in completely resected stage IB and II non-small-cell lung cancer: Updated survival analysis of JBR-10. J Clin Oncol 2010;28:29-34. |
[Table 1], [Table 2]