Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 18  |  Issue : 6  |  Page : 1733-1737

Stromal microenvironment namely angiogenesis, tumor-infiltrating lymphocytes, and matrix metalloproteinase in invasive breast carcinoma: Do they have a prognostic role?


1 Department of Pathology, North Delhi Medical College, Hindu Rao Hospital, Delhi, India
2 Division of Cytopathology, ICMR-National Institute of Cancer Prevention and Research, Noida, Uttar Pradesh, India

Date of Submission15-Jan-2021
Date of Decision27-Jan-2021
Date of Acceptance27-Jan-2021
Date of Web Publication10-Sep-2022

Correspondence Address:
Ruchika Gupta
Scientist-D, Division of Cytopathology, ICMR-National Institute of Cancer Prevention and Research, I-7, Sector-39, Noida - 201 301, Uttar Pradesh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.jcrt_95_21

Rights and Permissions
 > Abstract 


Background: The role of stromal microenvironment in growth, invasiveness, and metastatic potential of breast carcinoma (BC) is being recognized increasingly, both to predict prognosis and as potential therapeutic targets. The present study aimed to evaluate the correlation of angiogenesis, tumor-associated lymphocytes, and stromal CD10 expression with clinicopathologic parameters.
Materials and Methods: This study included 100 consecutive cases of invasive BC undergoing modified radical mastectomy. Relevant clinical details, pathological grade, lymph nodal status, and clinical stage were noted. Paraffin-embedded sections were subjected to immunohistochemistry for CD34, CD20, CD45RO, and CD10. Microvessel density (MVD), tumor-associated lymphocytes, and stromal CD10 expression were estimated from these sections. Statistical analysis was done using nonparametric tests to correlate the clinic-pathologic features with each of these parameters.
Results: MVD was found to be significantly higher in Grade III, node-positive cases, and higher stage breast cancers (P < 0.05). The number of T-lymphocytes was higher in node-positive cases, while B-lymphocytes were lower in number in higher grade tumors. CD10 expression showed a significant positive association with tumor grade, nodal status, and stage (P < 0.05 for each).
Conclusion: This study demonstrates that changes in stromal microenvironment of BC such as MVD, tumor-associated lymphocytes, and stromal CD10 expression correlate with the clinicopathological parameters and hence may be exploited as prognostic markers or therapeutic targets, based on further larger studies.

Keywords: Breast cancer, CD10, lymphocytes, microenvironment, stroma


How to cite this article:
Srivastava G, Pant L, Kudesia M, Singh S, Gupta R. Stromal microenvironment namely angiogenesis, tumor-infiltrating lymphocytes, and matrix metalloproteinase in invasive breast carcinoma: Do they have a prognostic role?. J Can Res Ther 2022;18:1733-7

How to cite this URL:
Srivastava G, Pant L, Kudesia M, Singh S, Gupta R. Stromal microenvironment namely angiogenesis, tumor-infiltrating lymphocytes, and matrix metalloproteinase in invasive breast carcinoma: Do they have a prognostic role?. J Can Res Ther [serial online] 2022 [cited 2022 Dec 3];18:1733-7. Available from: https://www.cancerjournal.net/text.asp?2022/18/6/0/355659




 > Introduction Top


Breast carcinoma (BC) is the most common malignancy in females worldwide as well as in India.[1] Given the high morbidity and mortality due to diagnosis in late stages, research has focused on unearthing newer prognostic markers and therapeutic targets, especially based on tumor stroma interaction.[2]

Tumors require neovascularization for their growth as well as metastasis to distant sites.[3] Angiogenesis is most often quantified using microvessel density (MVD) from CD34 or other vascular marker-stained sections. Studies have emphasized the role of angiogenesis in genesis, metastasis, and prognosis of BC.[3],[4] Tumor-associated lymphocytes, both T- and B-cells are increasingly been shown to be involved in host-tumor relationship in many solid tumors. Few studies have shown the presence of lymphocytes as a good prognostic indicator in BC.[5] On the other hand, others have suggested the role of lymphocytes in potentiating tumorigenesis and hence considered as a bad prognostic factor.[6]

Matrix metalloproteinases (MMPs), which facilitate invasiveness and metastatic potential of a tumor through basement membrane degradation, have been evaluated for their role as the prognostic factors in solid tumors including BC.[7] CD10 is a zinc-dependent peptidase which acts as metalloproteinase and degrades variety of bioactive peptides. Occasional studies have suggested that there is upregulation of CD10 in BC.[7]

However, the existing literature has conflicting reports of the correlation of angiogenesis, inflammatory cells with other clinicopathological prognostic factors.[7],[8],[9],[10] Only a few studies have studied significance of inflammatory cells, angiogenesis, and MMP in BC simultaneously.[6],[11]

The present study aimed at studying the role of stroma in BC progression by evaluating MVD, T, and B lymphocytes and CD10 expression in the stroma of BC tissues and to correlate these with clinicopathologic parameters namely histological grade, tumor, node, metastasis (TNM) stage, and axillary lymph node status.


 > Materials and Methods Top


This was a retrospective study including 100 consecutive cases of invasive BC that underwent modified radical mastectomy at our hospital. Clinical details of all cases including age, gender, laterality, and size of tumor were noted. Histopathological sections were reviewed for detailed morphological features: histological type of tumor, Nottingham modification of Bloom–Richardson grading (BR grade), nodal metastasis, Nottingham prognostic index (NPI), and pathological tumor stage (pTNM).

Immunohistochemistry was performed on carefully selected representative tumor sections for CD34 (MVD), CD45 RO (T-cell), CD20 (B-cell), and CD10 (MMPs) as per standard protocols using the LSAB technique. All the primary antibodies as well as the detection system was procured from Biogenex, USA. MVD was assessed from CD34-stained sections at low magnification (×10) in the area with highest vascular density (hotspot). Any brown staining endothelial cell that was clearly separated from adjacent vessels was considered a single microvessel and counted. The presence of lumen was not required for classification of a structure as a microvessel, as per standard criteria.[12] An average of three “hotspots” was taken to calculate the mean MVD in each case.

For quantitation of tumor-associated lymphocytes, representative sections stained for both T cells and B cells were subjected to manual counting of cells in at least ten different high power fields (HPF) (per 0.033 mm2). Areas of necrotic foci were avoided. The results were expressed as mean number of T and B cells per HPF.

Stromal CD10 expression was graded as 1, 2, and 3 considering myoepithelial cell staining as Grade 3. Grade 2 and 3 staining was considered as positive. Total stromal positivity of >30% was considered as CD10 positive. For statistical analysis, tumor stage was grouped as Stage II and III, without further subclassification.

Nonparametric Mann–Whitney test was applied to compare the MVD, mean T and B lymphocytes with clinic-pathologic parameters. For comparison of CD10 expression with clinicopathologic parameters, Chi-square test was applied. A P < 0.05 was considered to be significant.


 > Results Top


The mean age of patients in our study was 47.1 years, with maximum number of cases in the age group of 40–49 years. All the patients were females. Of the 100 cases, 93 were classified as invasive ductal carcinoma (IDC) [not otherwise specified (NOS) type, [Figure 1]a, while three were mucinous, two papillary carcinoma, and one each was adenoid cystic carcinoma and carcinosarcoma. The mean tumor size was 4.33 cm (range: 1–12 cm). Tumor grading as per the Nottingham modification of BR grade system was applied to 93 cases of IDC-NOS. Six cases (6.4%) were categorized as Grade I tumor, 51 (54.8%) as Grade II, and 36 (38.7%) as Grade III tumors. The cases classified as mucinous, papillary, adenoid cystic, and carcinosarcoma were not graded. TNM staging revealed 16 cases (17.2%) as Stage IIA, 47 (50.5%) in stage IIB, 21 (22.5%) as Stage IIIA, and nine (9.6%) as Stage IIIB tumors. None of the cases were diagnosed at Stage I. Hence, 63 cases were Stage II, while thirty cases were Stage III at the time of surgery. Eight (8.6%) cases conformed to NPI Group I, 40 cases (43%) in Group II, and rest 45 cases (48.4%) in Group III. The seven cases where BR grading was not applicable were not subjected to calculation of NPI.
Figure 1: A panel of photomicrographs showing invasive ductal carcinoma – not otherwise specified type (a, H and E, ×100) with high microvessel density in a CD34-stained area (b, ×100). Tumor-infiltrating lymphocytes (c, H and E, ×400) highlighted by CD45RO staining for T lymphocytes (d, ×100) and CD20 for B lymphocytes (e, ×100). One of the cases displays strong stomal positivity for CD10 (f, ×100)

Click here to view


The mean MVD [Figure 1]b of all cases was 80.26 ± 30.47, ranging from 30 to 150. For cases of IDC-NOS, MVD varied between 30 and 150 with a mean of 82.05 ± 29.12, while MVD of three cases of mucinous carcinoma was 30, 40, and 42. Similarly, MVD of the cases reported as papillary, adenoid cystic, and carcinosarcoma was 54, 56, 65, and 70, respectively.

Tumor-associated T lymphocytes [Figure 1]c and [Figure 1]d ranged between 11 and 230 per HPF with an average of 118.44 ± 24.95 per HPF. The mean T-lymphocytes varied from 118.4 ± 42.9 in IDC-NOS to 81 per HPF in mucinous carcinoma. On the other hand, density of B-lymphocytes [Figure 1]e varied from 4 to 68 per HPF, mean being 44.3 ± 21.5 per HPF. The average B-lymphocytes in cases of IDC-NOS was 43.2 ± 21.2 per HPF.

Stromal CD10 expression was found to be strong in 70% of cases [Figure 1]f, while weak positivity was noted in rest 30% of tumors. Of the 93 cases of IDC-NOS, 71 (76.3%) showed strong stromal CD10 positivity, while 22 cases (23.6%) were considered as negative. The two cases of papillary carcinoma also showed weak CD10 expression in stromal cells. Mucinous carcinoma and carcinosarcoma cases were negative for stromal CD10 expression.

Microvessel density and clinicopathologic parameters

The mean MVD ranged from 77.0 in cases categorized as BR Grade I to 80 in Grade II and 84.2 in Grade III tumors. The difference in MVD was statistically significant between Grade I and III (P = 0.04); however, the same was not true for difference between Grade I and II or II and III. A significant difference was noted in mean MVD between node-negative and node-positive cases of IDC (P = 0.01), as shown in [Table 1].
Table 1: Association of microvessel density, lymphocytes, and CD10 expression with clinicopathologic parameters

Click here to view


Mean MVD was significantly higher in TNM Stage III cases compared to Stage II cases (P = 0.035), while no significant correlation was found between MVD and NPI.

Tumor-associated lymphocytes and various clinic-pathologic features

The mean number of T-lymphocytes was higher in BR Grade II and III tumors compared to Grade I, though the difference was not statistically significant [Table 1]. Node-positive cases have significantly higher mean T cells than node-negative cases (P = 0.039). The difference in mean T-cell number between NPI Grade I and III was found to be significant (P = 0.03). Although the mean number of T-cells was higher in Stage III compared to Stage II tumors, the difference was not significant.

In comparison to T lymphocytes, a higher number of B cells were associated with lower tumor grade, stage, and NPI status [Table 1]. However, no association was found between number of B lymphocytes and nodal involvement by BC.

CD10 expression in various clinic-pathologic groups of breast carcinoma

Among the cases of IDC-NOS, CD10 expression was found to be associated with higher tumor grade, advanced stage, and nodal metastasis. CD10 positivity was higher in cases of NPI Group III compared to Grade I [Table 1].


 > Discussion Top


Although tumor grade, stage, lymph node status, type of cancer, hormone receptor status, and Her-2 positivity are accepted prognostic factors for BC, newer markers are being explored to facilitate the application of personalized medicine and targeted therapy in BC patients. In this regard, recent interest has been focused on the role of tumor microenvironment on biologic behavior and the role of stromal elements on tumor progression.[13] The tumor microenvironment, comprised mesenchymal cells and those of hematopoietic origin as well as noncellular components, is a source of epigenetic regulation of BC.[14] The cellular component of tumor microenvironment includes fibroblasts, myofibroblasts, mesenchymal stem cells, adipocytes, endothelial cells, lymphoid cells, and myeloid cells.[15]

Angiogenesis has long been recognized as an important factor for growth, invasion, and metastasis of a variety of tumors including BC.[16] MVD, assessed using endothelial markers such as CD31, CD34, or CD105, is now the gold standard for assessing angiogenesis. In our study, significantly higher MVD was seen in Grade III tumors, those with nodal metastasis, and Stage III compared to Stage II cases. This is in consonance with the earlier studies evaluating MVD in BC.[17],[18] A significant increase in angiogenesis has also been demonstrated during the progression of ductal proliferations in breast from atypical ductal hyperplasia to carcinoma – in situ to invasive carcinoma.[19]

Tumor-infiltrating lymphocytes (TILs) in BC have been shown to be associated with favorable outcome in long-term follow-up.[5] Majority of the studies have demonstrated that TILs are predominantly composed of T lymphocytes, while B cells constitute a minor population.[20] This is also reflected in our results where the mean number of T lymphocytes was 118.4 compared to 43.2 for B cells. Significantly, a higher number of T cells were seen in node-positive cases in our study and in cases with higher NPI. Although the number of T lymphocytes was higher in Grade III tumors, the difference did not reach statistical significance. Earlier studies have found increasing infiltration by mononuclear cells in higher grade tumors.[6] TILs have also been shown to be associated with nodal positivity in BC.[20] In contrast, the mean number of B lymphocytes was found to be lower in higher grade, stage, and cases in NPI Group III. Since B-cells usually constitute a minority of TILs, studies evaluating their association with clinic-pathologic parameters are fewer in the existing literature. A study by Mahmoud et al. showed that a number of B cells were higher in the stroma at a distance from the tumor. The authors also demonstrated a positive correlation of total CD20 + cells with higher tumor grade, hormone receptor negativity, and basal phenotype of tumor cells. Univariate and multivariate analysis in their study showed that total CD20 + count was associated with better survival and longer disease-free interval.[21] In contrast, a recent study showed that B lymphocytes were not associated with histologic grade or clinical stage of the tumor in BC patients.[22]

CD10, also known as neprilysin or common acute lymphoblastic leukemia/lymphoma antigen, is a zinc-dependent metalloproteinase produced by myofibroblasts in the stroma. CD10 has been identified in stromal cells of normal breast as well as in various tumors such as gastric, lung, prostate, colorectal, and breast.[23],[24] The proteolytic activity of CD10 is hypothesized to contribute to the invasiveness and metastatic potential of tumors with CD10-positive stromal cells. CD10 expression has been associated with nodal metastasis and vascular invasion in colorectal carcinoma,[23] advanced stage tumors in nasopharyngeal carcinoma[25] as well as in melanoma.[26] In BC, CD10 expression by stromal cells has been shown to be associated with nodal metastasis and high tumor grade.[24] However, the results have been conflicting since some authors did not find correlation of CD10 expression with lymph node involvement in BC.[7],[10] In the present study, we found CD10 expression to be significantly associated with higher grade tumors, advanced stage, nodal metastasis, and higher NPI (P < 0.05 for all). These are in consonance with the earlier results of Kim et al.[27] Some authors have also demonstrated the association of CD10 expression with reduced long-term disease-specific survival.[7],[27] A recent study reported stromal CD10 expression to be associated with lymph node involvement and higher grade tumors. In addition, CD10 positivity in stromal cells was associated with cancer stem cell phenotype assessed by CD44 and ALDH1.[28] The authors suggested a role of cancer stem cells in the induction of expression of CD10 by stromal cells in the tumor, though further studies are required to confirm this hypothesis. This variation in the results of various studies may be partly attributed to the criteria utilized for considering a case as positive for CD10 (10% or 30% strong positive staining).

CD10 expression is also thought to have a role in BC therapeutics. In order to maximize the efficacy with simultaneous reduction in toxicity of anticancer drugs, peptide prodrugs are being developed that may be cleaved by the peptidases in the tumor microenvironment enabling direct action on the tumor cells without much action on the healthy tissues. One such prodrug, CPI-004Na, is cleaved by CD10 into active drug at a higher potency than systemically delivered drug.[29] Hence, it is likely that, in future, CD10 immunostaining may be required for assistance in therapeutic decision-making for BC patients.


 > Conclusion Top


To the best of our knowledge, none of the earlier studies have attempted to evaluate the various factors of tumor microenvironment in BC together. Ours is the first such study that assessed angiogenesis, tumor-associated lymphocytes, and CD10 metalloproteinase in breast cancer for their association with vital clinic-pathologic parameters. Our results indicate that these parameters could serve as prognostic markers or therapeutic targets. Further larger prospective studies are required to confirm and validate our results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394-424.  Back to cited text no. 1
    
2.
Anne VS, Thiery JP. Host microenvironment in breast cancer development: Epithelial mesenchymal transition in breast carcinoma development. Breast Cancer Res 2003;5:101-6.  Back to cited text no. 2
    
3.
Kim JB, Stein R, Mike J, O'Hare. Tumour stromal interaction in breast cancer: The role of stroma in tumourigenesis. Tumor Biol 2005;26:173-85.  Back to cited text no. 3
    
4.
Bryan PS, Miller KD. Angiogenesis of breast cancer. J Clin Oncol 2005;23:1782-90.  Back to cited text no. 4
    
5.
DeNardo DG, Coussens LM. Inflammation and breast cancer. Balancing immune response: Crosstalk between adaptive and innate immune cells during breast cancer progression. Breast Cancer Res 2007;9:212.  Back to cited text no. 5
    
6.
Murri AM, Hilmy M, Bell J, Wilson C, McNicol AM, Lannigan A, et al. The relationship between the systemic inflammatory response, tumour proliferative activity, T-lymphocytic and macrophage infiltration, microvessel density and survival in patients with primary operable breast cancer. Br J Cancer 2008;99:1013-9.  Back to cited text no. 6
    
7.
Makretsov NA, Hayes M, Carter BA, Dabiri S, Gilks CB, Huntsman DG. Stromal CD10 expression in invasive breast carcinoma correlates with poor prognosis, estrogen receptor negativity and high grade. Mod Pathol 2007;20:84-9.  Back to cited text no. 7
    
8.
Bujor IS, Cioca A, Ceauşu RA, Veaceslav F, Nica C, Cîmpean AM, et al. Evaluation of vascular proliferation in molecular subtypes of breast cancer. In Vivo 2018;32:79-83.  Back to cited text no. 8
    
9.
Shrivastav S, Bal A, Singh G, Joshi K. Tumor Angiogenesis in breast cancer: Pericytes and maturation does not correlate with lymph node metastasis and molecular subtypes. Clin Breast Cancer 2016;16:131-8.  Back to cited text no. 9
    
10.
Iwaya K, Ogawa H, Izumi M, Kurodo M, Mukai K. Stromal expression of CD10 in invasive breast carcinoma: A new predictor of clinical outcome. Virchow Arch 2002;440:589-93.  Back to cited text no. 10
    
11.
Oshi M, Newman S, Tokumaru Y, Yan L, Matsuyama R, Endo I, et al. Intra-tumoral angiogenesis is associated with inflammation, immune reaction and metastatic recurrence in breast cancer. Int J Mol Sci. 2020;21:6708.  Back to cited text no. 11
    
12.
Aggarwal D, Srivastava G, Gupta R, Pant L, Krishan G, Singh S. Angiogenesis in non-Hodgkin's lymphoma: An intercategory comparison of microvessel Density. ISRN Hematol 2012;2012:943089.  Back to cited text no. 12
    
13.
Hanahan D, Coussens LM. Accessories to the crime: Functions of cells recruited to the tumor microenvironment. Cancer Cell 2012;21:309-22.  Back to cited text no. 13
    
14.
Pattabiraman DR, Weinberg RA. Tackling the cancer stem cells what challenges do they pose? Nat Rev Drug Discov 2014;13:497-512.  Back to cited text no. 14
    
15.
McCuaig R, Wu F, Dunn J, Rao S, Dahlstrom JE. The biological and clinical significance of stromal-epithelial interactions in breast cancer. Pathology 2017;49:133-40.  Back to cited text no. 15
    
16.
Uzzan B, Nicolas P, Cucherat M, Perret GY. Microvessel density as a prognostic factor in women with breast cancer: A systematic review of the literature and meta-analysis. Cancer Res. 2004;64:2941-55.  Back to cited text no. 16
    
17.
Pyakurel D, Karki S, Agrawal C. A study on microvascular density in breast carcinoma. J Pathol Nepal 2014;4:570-5.  Back to cited text no. 17
    
18.
Safwat MD, Habib F, Elayat A, Oweiss N, Reffat S, Algaidi S. Morphometric and immunohistochemical study of angiogenic marker expressions in invasive ductal carcinomas of the human breast. Folia Morphol 2009;68:144-55.  Back to cited text no. 18
    
19.
Carpenter PM, Chen WP, Mendez A, McLaren CE, Su MY. Angiogenesis in the progression of breast ductal proliferations. Int J Surg Pathol 2011;19:335-41.  Back to cited text no. 19
    
20.
Macchetti AH, Marana HR, Silva JS, de Andrade JM, Ribeiro-Silva A, Bighetti S. Tumor-infiltrating CD4+ T lymphocytes in early breast cancer reflect lymph node involvement. Clinics (Sao Paulo) 2006;61:203-8.  Back to cited text no. 20
    
21.
Mahmoud SM, Lee AH, Paish EC, Macmillan RD, Ellis IO, Green AR. The prognostic significance of B lymphocytes in invasive carcinoma of the breast. Breast Cancer Res Treat 2012;132:545-53.  Back to cited text no. 21
    
22.
Helal TE, Ibrahim EA, Alloub AI. Immunohistochemical analysis of tumor-infiltrating lymphocytes in breast carcinoma: Relation to prognostic variables. Indian J Pathol Microbiol 2013;56:89-93.  Back to cited text no. 22
[PUBMED]  [Full text]  
23.
Huang WB, Zhou XJ, Chen JY, Zhang LH, Meng K, Ma HH, et al. CD10-positive stromal cells in gastric carcinoma: Correlation with invasion and metastasis. Jpn J Clin Oncol. 2005;35:245-50.  Back to cited text no. 23
    
24.
Makretsov NA, Hayes M, Carter BA, Dabiri S, Gilks CB, Huntsman DG. Stromal CD10 expression in invasive breast carcinoma correlates with poor prognosis, estrogen receptor negativity, and high grade. Mod Pathol. 2007;20:84-9.  Back to cited text no. 24
    
25.
Braham H, Trimeche M, Ziadi S, Mestiri S, Mokni M, Amara K, et al. CD10 expression by fusiform stromal cells in nasopharyngeal carcinoma correlates with tumor progression. Virchows Arch 2006;449:220-4.  Back to cited text no. 25
    
26.
Bilalovic N, Sandstad B, Golouh R, Selak I, Torlakovic EE. CD10 protein expression in tumor and stromal cells of malignant melanoma is associated with tumor progression. Mod Pathol 2004;17:1251-8.  Back to cited text no. 26
    
27.
Kim HS, Kim GY, Kim YW, Park YK, Song JY, Lim SJ. Stromal CD10 expression and relationship to the E-cadherin/beta-catenin complex in breast carcinoma. Histopathology 2010;56:708-19.  Back to cited text no. 27
    
28.
Louhichi T, Saad H, Dhiab MB, Ziadi S, Trimeche M. Stromal CD10 expression in breast cancer correlates with tumor invasion and cancer stem cell phenotype. BMC Cancer 2018;18:49.  Back to cited text no. 28
    
29.
Pan C, Cardarelli PM, Nieder MH, Pickford LB, Gangwar S, King DJ, et al. CD10 is a key enzyme involved in the activation of tumor-activated peptide prodrug CPI-0004Na and novel analogues: Implications for the design of novel peptide prodrugs for the therapy of CD10+ tumors. Cancer Res 2003;63:5526-31.  Back to cited text no. 29
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  >Abstract>Introduction>Materials and Me...>Results>Discussion>Conclusion>Article Figures>Article Tables
  In this article
>References

 Article Access Statistics
    Viewed357    
    Printed118    
    Emailed0    
    PDF Downloaded16    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]