ent step in the protocol. Deparaffinization: This has been performed previously after the sections were dried for 1 hour at 60˚C. Pretreatments: [Heat induced epitope retrieval in microwave-oven (MWO)].

2.4. Scoring System

The criterion for positive immunoreaction is dark brown intracellular (cytoplasmic) precipitate for VEGF, or at the cell membrane for HER-2/neu and in the nucleus for ER and PR. The proportion of the staining was assessed by counting the percentage of positive cells in 100 malignant cells at objective 40 total magnifications, each sample was scanned for five fields randomly with a high power magnification (144). Qualitative assessment: Faint staining pattern, whether cytoplasmic, membranous or nuclear, that only could be detected by using higher magnification (objective 40), while strong staining pattern, easily seen by low magnification (objective 4).

a) VEGF Scoring System (Table 1)

b) HER-2/neu Scoring System (Table 2)

c) Estrogen and Progesterone Receptors Scoring System [20] :

A total score (TS) = Sum of Proportion Score (PS) and Intensity Score (IS). A positive result for both ER and PR is defined as TS ≥ 3 (Table 3, Table 4).

Table 1. VEGF Scoring system [18] .

Table 2. Herceptest Scoring system [19] .

HER-2/neu protein overexpression assessment includes [19] : Score 0 = negative; Score 1+ = negative; Score 2+ = equivocal (need FISH study); Score 3+ = positive.

Table 3. ER and PR Proportion scoring system (PS).

Table 4. ER and PR Intensity scoring system (IS).

2.5. Statistical Analysis

All biopsies were classified into three grades: Grade I, Grade II and Grade III, according to the modified Bloom Richardson Grading System [21] . Statistical analyses of all results were performed by the help of SPSS software statistical package (version 15) using Chi Square test, P value at level of significance <0.05 and correlation regression test (R at a significant level of 0.3).

3. Results

Forty four (84.62%) of the fifty two cases included in this study were of ductal carcinoma while 8 cases (15.38%) were of lobular type. The detection rate of VEGF, HER-2/neu, ER and PR were 59.62%, 36.96%, 34.62% and 36.54% respectively. The positivity of VEGF were 0% in normal breast tissue, 20% in the benign and 59.62% in the malignant tissues with a significant difference (P < 0.05) (Table 5) in immunoexpression as it also occur between ductal and lobular type carcinoma, but not significantly different among tumor grades, different tumor sizes, axillary lymph node involvement and age of the patients. However, VEGF was positively correlated with tumor grade (0% for grade I, 50% grade II, 65.79% grade III), tumor size if excluding in situ component, also with positive nodal involvement (Table 5), also with HER-2/neu (14 out of 17 HER2 positive cases were VEGF positive while 14 out of 29 HER2 negative cases were VEGF positive) (Table 6), but negatively correlated with ER and PR, as explained: (8 out of 18 ER positive cases were VEGF positive while 23 out of 34 ER negative cases were VEGF positive) (Table 7), (10 out of 19 PR positive cases were VEGF positive while 21 out of 33 PR negative cases were VEGF positive) without a significant difference (P > 0.05) and these show the most unfavorable biopathological profile.

4. Discussion

Breast cancer is a multifactorial disease, the genetic alterations lead to the transformation of normal cells into cancer cells. Human breast cancer is an angiogenesis-dependent tumor that initially depends on estrogens for development and progression [22] . The biology of breast carcinoma remains poorly understood as the knowledge about individual prognostic factors provides limited information [23] . Today, most pathologists considered IHC evaluation of ER/PR receptors as well as c-erbB-2, VEGF and Ki-67 proliferation indices as essential parameters in selecting the line of treatment of breast cancer patient in addition to the traditional tumor/no- dal/metastasis staging variables [24] . Thus, the current study represents a forward step toward understanding the possible role of VEGF during breast cancer development, invasion, and metastasis, moreover, determining ER, PR and HER-2/neu overexpression and their possible correlation to VEGF overexpression in different clinicopathological parameters of breast carcinoma in a series of Iraqi women to select the most appropriate treatment for each patient. The current study demonstrated that although completely absent expression from normal breast tissue and relatively little expression in benign breast lesions, conversely there was a significant overexpression of VEGF among the 52 investigated breast carcinoma (P value < 0.05). The results have clarified that 59.62% of breast cancer cases were expressing VEGF immunostaining in their histological sections (Table 5). Our results are near to those reported by other investigators [9] [11] [22] [25] [26] , without any significant difference between our results and these results (P > 0.05), while they are higher than those reported by some others [27] -[29] .

Immunoexpression of VEGF for ductal carcinomas was higher than that of infiltrating lobular carcinomas (65.91% versus 25%) with a significant difference (P < 0.05) (Table 5). Thus, supporting the view that such tumors represent a defined subtype of breast carcinoma. The current study revealed 100% of pure DCIS and DCIS with invasive component were VEGF positive. It looks that VEGF may play a role in cancer progression from CIS to invasive form, and may influence aggressiveness indirectly through its effect on angiogenesis, furthermore, it acts as an autocrine survival for the tumor cells themselves as documented by many researches [30] [31] . These results are supporting the hypothesis that VEGF plays important role in initiation and progression of ductal carcinomas. Although positive correlation with higher grades was detected, there is no significant difference among these grades (R > 0.3, P > 0.05). This fact has been documented by many investigators [9] [13] [32] -[34] .The unexpected findings may be due to disproportionate total number of cases in grade II and III (12 versus 38) which may interrupt the significant association (Table 5). Another explanation is that as the tumor advances, other biological changes may occur that reduce the requirement for continued VEGF signaling, these changes reflect the aggressive behavior of tumor and the reduced degree of tumor differentiation. It looks that

Table 5. Relationship between VEGF immunoexpression and clinicopathological parameters of breast carcinoma.

the detection rate of VEGF is increasing as the size of tumor increased (R > 0.3) without a significant difference (P > 0.05) among different tumor sizes if in situ components were excluded from the data (Table 5).

Similar findings found by other researches [17] [18] , but many papers documented that there is a negative correlation between VEGF expression and size of tumor [26] [32] [35] [36] . This might be attributed to the effect of hypoxia, the most important stimulator of VEGF production, which becomes more profound as the size of the tumor increases. VEGF immunoexpression is higher in node positive breast cancer than in node negative without a significant difference between them (Table 5). This finding against what was suggested by one study in

Table 6. Coexpression of VEGF and HER-2/neu in relation to their separate and neither expression in breast carcinoma.

Table 7. Coexpression of VEGF and ER in relation to their separate and neither expression in breast carcinoma.

(2003) [26] , but agreed with what was reported by others [23] [33] [37] -[40] and this may be attributed to the aggressive behavior of node positive breast cancer. The results of this study revealed that there was no significant difference in VEGF immunoexpression among different age groups, this may be corresponding to the natural frequency of breast cancer. HER-2/neu positivity was documented in 14 (50%) out of 28 cases with positive VEGF immunoexpression, there is positive correlation with a significant difference (Table 6). This finding agreed with many studies [41] -[43] . ER positivity was seen in 8 (25.81%) out of 31 cases with positive VEGF immuno-expression, without a significant difference (Table 7). This finding agreed with many authors [10] [22] [29] [41] [42] [44] -[47] .

The above authors proposed several possible explanations, including limitation of tumor growth by vascular compression, interstitial pressure, and necrosis; a lack of specific receptors for soluble ER isoforms in this subgroup of high VEGF expressing tumors; overexpression of endo-genous angiogenic inhibitors; or the distortion of results due to relatively small number of patients in this subgroup which also may be a factor affecting the study results. VEGF expression may be regulated by estrogen and has been correlated with ER-negative status and associated with response to anti estrogen therapy. PR positivity was seen in 10 (32.26%) out of 31 cases with positive VEGF immunoexpression, without a significant difference. This finding agreed with some researches [41] [44] . The absence of such association is anticipated because breast cancers express numerous angiogenic factors that act in concert to generate the tumor vasculature as it is interacting with hormonal receptors. These differences may also reflect the effect of varying therapeutic regimens on the interaction with steroid receptors.

5. Conclusions

In conclusion, normal and benign breast tissues did not express a significant immunohistochemical level of VEGF. VEGF overexpression is a hallmark of bad prognosis as it was associated with worse biopathological parameters, such as tumor size. VEGF immunoexpression was positively correlated with HER-2/neu, while inversely correlated with ER and PR immunoexpression. Incorporation of these biomarkers with other factors into a prognostic index will more accurately predict clinical outcome and determine the effect of anti cancer therapy.

Large prospective clinical studies with a longer duration of follow-up and studying the survival rates will provide a better insight and validate our findings and are needed to better clarify the prognostic role of VEGF in breast cancer. Considering further studies including DNA/mRNA and protein levels by FISH or PCR techniques to confirm the molecular basis of these genes alteration and to reveal which members of the VEGF family might possibly be useful in identifying those patients who will benefit most from anti-VEGF strategies.

Competing Interests

The author declared that she had no competing interests.

Cite this paper

MaisAlmumen, (2015) Immunohistochemical Expression of VEGF in Relation to Other Pathological Parameters of Breast Carcinoma. Journal of Cancer Therapy,06,811-820. doi: 10.4236/jct.2015.69089

References

  1. 1. Singhal, H., Gohel, M.S., Kaur, K. and Thomson, S. (2008) Breast Cancer Evaluation. eMedicine Journal Specialty, 2-10.

  2. 2. Folkman, J. and Klagsbrun, M. (1987) Angiogenic Factors. Science, 235, 442-447.
    http://dx.doi.org/10.1126/science.2432664

  3. 3. Folkman, J. (1989) What Is the Evidence that Tumors Are Angiogenesis Dependent? Journal of the National Cancer Institute, 82, 4-6.
    http://dx.doi.org/10.1093/jnci/82.1.4

  4. 4. Drovak, H.F., Brown, L.F., Detmar, M. and Dvorak, A.M. (1995) Vascular Permeability Factor/Vascular Endothelial Growth Factor, Microvascular Hyperpermeability, and Angiogenesis. American Journal of Pathology, 146, 1029-1039.

  5. 5. Ferrara, N. and Alitalo, K. (1999) Clinical Application of Angiogenic Growth Factors and Their Inhibitors. Nature Medicine, 5, 1359-1364.
    http://dx.doi.org/10.1038/70928

  6. 6. Nicosia, R.F. (1998) What Is the Role of Vascular Endothelial Growth Factor-Related Molecules in Tumor Angiogenesis? The American Journal of Pathology, 153, 11-16.
    http://dx.doi.org/10.1016/S0002-9440(10)65539-3

  7. 7. Jung, L., Balan, B.J., Rózewska, E.S., Wynimko, J.C., Siwicki, A.K., Sommer, E., et al. (2007) Clinical Immunology VEGF in Circulating Blood of Patients Treated with Enoxaparine after Orthopaedic Surgery. Central European Journal of Immunology, 32, 61-65.

  8. 8. Ferrara, N. (1999) Molecular and Biological Properties of Vascular Endothelial Growth Factor. Journal of Molecular Medicine, 77, 527-543.
    http://dx.doi.org/10.1007/s001099900019

  9. 9. Linderholm, B., Tavelin, B. and Grankvist, K. (1998) Vascular Endothelial Growth Factor is of High Prognostic Value in Node-Negative Breast Carcinoma. Journal of Clinical Oncology, 16, 3121-3128.

  10. 10. Linderholm, B., Lindh, B., Tavelin, B., Grankvist, K. and Henriksson, R. (2000) p53 and Vascular-Endothelial-Growth-Factor (VEGF) Expression Predicts Outcome in 833 Patients with Primary Breast Carcinoma. International Journal of Cancer, 89, 51-62.
    3.0.CO;2-8>http://dx.doi.org/10.1002/(SICI)1097-0215(20000120)89:1<51::AID-IJC9>3.0.CO;2-8

  11. 11. Toi, M., Hoshina, S. and Takayanagi, T. (1994) Association of Vascular Endothelial Growth Factor Expression with Tumor Angiogenesis and with Early Relapse in Primary Breast Cancer. Japanese Journal of Cancer Research, 85, 1045-1049.
    http://dx.doi.org/10.1111/j.1349-7006.1994.tb02904.x

  12. 12. Toi, M., Inada, K., Suzuki, H. and Tominaga, T. (1995) Tumor Angiogenesis in Breast Cancer. Breast Cancer Research and Treatment, 36, 193-204.
    http://dx.doi.org/10.1007/BF00666040

  13. 13. Valkovic, T., Dobrila, F., Melato, M., Sasso, F., Rizzardi, C. and Jonjic, N. (2002) Correlation between Vascular Endothelial Growth Factor, Angiogenesis, and Tumor-Associated Macrophages in Invasive Ductal Breast Carcinoma. Virchows Archiv, 440, 583-588.
    http://dx.doi.org/10.1007/s004280100458

  14. 14. Wang, F., Wei, L. and Chen, L. (2000) The Relationship between Vascular Endothelial Growth Factor, Microvascular Density, Lymph Node Metastasis and Prognosis of Breast Carcinoma. Chinese Journal of Pathology, 29, 172-175.

  15. 15. MacConmara, M., O’Hanlon, D.M., Kiely, M.J., Connolly, Y., Jeffers, M. and Keane, F.B. (2002) An Evaluation of the Prognostic Significance of Vascular Endothelial Growth Factor in Node Positive Primary Breast Carcinoma. International Journal of Oncology, 20, 717-721.
    http://dx.doi.org/10.3892/ijo.20.4.717

  16. 16. Ferrara, N. and Henzel, W.J. (1989) Pituitary Follicular Cells Secrete a Novel Heparin-Binding Growth Factor Specific for Vascular Endothelial Cells. Biochemical and Biophysical Research Communications, 84, 856-861.
    http://dx.doi.org/10.1016/0006-291x(89)92678-8

  17. 17. World Health Organization International Agency for Research on Cancer (2003) World Cancer Report, 335.

  18. 18. Apple, S.K., Hecht, J.R., Lewin, D.N., Jahromi, S.A., Grody, W.W. and Nieberg, R.K. (1999) IHC Evaluation of K-ras, P53, and Her2/neu Expression in Hyperplasic, Dysplastic, and Carcinomatous Lesions of the Pancreas: Evidence for Multistep Carcinogenesis. Human Pathology, 30, 123-129.
    http://dx.doi.org/10.1016/S0046-8177(99)90265-4

  19. 19. Truls, G., Kennet, W. and Jorgen, C. (2005) Analysis of Her2/neu Expression in Primary Bladder Carcinoma and Corresponding Metastases. Journal of Pathology, 95, 982-986.

  20. 20. (2011) Dakocytomation Catalog for Products and Services. Dako, Glostrup, 139-143.

  21. 21. Elston, C.W. and Ellis, I.O. (1991) Pathological Prognostic Factors in Breast Cancer. I. The Value of Histological Grade in Breast Cancer: Experience from a Large Study with Long-Term Follow-Up. Histopathology, 19, 403-410.
    http://dx.doi.org/10.1111/j.1365-2559.1991.tb00229.x

  22. 22. Gasparini, G., Toi, M., Miceli, R., Vermeulen, P.B., Dittadi, R., Biganzoli, E., et al. (1999) Clinical Relevance of VEGF and Thymidine Phosphorelase in Patients with Node-Positive Breast Cancer Treated with Either Adjuvant Chemotherapy or Hormone Therapy. The Cancer Journal from Scientific American, 5, 101-112.

  23. 23. Sorli, T., Perou, C.M., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., et al. (2001) Gene Expression Patterns of Breast Carcinomas Distinguishes Tumor Subclasses with Clinical Implication. Proceedings of the National Academy of Sciences of the United States of America, 98, 10869-10874.
    http://dx.doi.org/10.1073/pnas.191367098

  24. 24. Allred, D.C., Harvey, J.M., Berardo, M. and Clark, G.M. (1998) Prognostic and Predictive Factors in Breast Cancer by Immunohistochemical Analysis. Modern Pathology, 11, 155-168.

  25. 25. Granato, A.M., Nanni, O., Falcini, F., Folli, S., Mosconi, G., Paola, F.D., et al. (2004) bFGF and VEGF Serum Levels in Breast Cancer Patients and Healthy Women. Breast Cancer Research, 6, R38-R45.
    http://dx.doi.org/10.1186/bcr745

  26. 26. Li, J., Song, S.T., Jiang, Z.F., Liu, X.Q. and Yan, L.D. (2003) Significance of Microvascular Density and VEGF in Breast Cancer. Chinese Journal of Oncology, 25, 145-153.

  27. 27. Ihemelandu, C.U., Leffall, L.D., Naab, T.J. and Fredrieck, W.A. (2007) Expression of VEGF, p53 in Molecular Breast Cancer Subtypes of Pre-Menopausal African-American Women. Journal of Clinical Oncology, 25.

  28. 28. Matkowski, R., Gisterek, I., Suder, E., Lacko, A., Szelachowska, J., Ramsey, D. and Kornafel, J. (2006) Correlation between VEGF and C-Met Expressions in Breast Carcinoma. Journal of Clinical Oncology, 24.

  29. 29. Elli, I., Charchanti, A., Briasoulis, E., Karavasilis, V., Batsis, C., Pavlidis, N., et al. (2002) The Prognostic Value of Vascular Endothelial Growth Factor (VEGF) in Invasive Breast Cancer: Correlation with Microvessel Density (MVD), ER, PgR, p53 and Proliferative Associated Indices (Ki-67, PCNA). Electronic Journal of Pathology and Histology, 8, 024-007.

  30. 30. Harmey, J.H. and Bouchier-Hayes, D. (2002) VEGF, a Survival Factor for Tumour Cells: Implications for Anti-Angiogenic Theraoy. Bioassays, 24, 280-283.
    http://dx.doi.org/10.1002/bies.10043

  31. 31. Bachelder, R.E., Crago, A., Chung, J., Wendt, M.A., Shaw, L.M., Robinson, G. and Mercurio, A.M. (2001) VEGF Is an Autocrine Survival Factor for Neuropilin-Expressing Breast Carcinoma Cells. Cancer Research, 61, 5736-5776.

  32. 32. Halimi, M., Vahedi, A. and Mostafapour, E.K. (2012) Association of VEGF with Regional Lymph Node Metastasis in Breast IDC. Journal of Medical Sciences, 12, 18-23.
    http://dx.doi.org/10.3923/jms.2012.18.23

  33. 33. Xu, W., Wang, G., Zou, Y., Song, J., Yang, X. and Wang, W. (2007) VEGF Expression in IDC of Breast. Chinese Journal of Cancer Research, 19, 56-59.
    http://dx.doi.org/10.1007/s11670-007-0056-y

  34. 34. Bolat, F., Kayaselcuk, F., Nursal, T.Z., Yagmurdur, M.C., Bal, N. and Demirhan, B. (2006) Microvessel Density, VEGF Expression, and Tumor Associated Macrophages in Breast Tumors. Journal of Experimental & Clinical Cancer Research, 25, 365-437.

  35. 35. Macolm, M., Deidre, O.M., James, K.M., Yvonne, C., Michael, J. and Keane, F.B. (2002) An Evaluation of the Prognostic Significance of VEGF in Node Positive Primary Breast Carcinoma. The Internet Journal of Oncology, 20, 717-721.

  36. 36. Gasparini, G., Toi, M., Gion, M., Verderio, P., Dittadi, R., Hanatani, M., et al. (1997) Prognostic Significance of VEGF Protein in Node-Negative Breast Carcinoma. Journal of the National Cancer Institute, 89, 139-186.
    http://dx.doi.org/10.1093/jnci/89.2.139

  37. 37. Hao, L., Zhang, C., Qiu, Y., Wang, L., Luo, Y., Jin, M., et al. (2007) Recombination of CXCR4, VEGF, and MMP-9 Predicting Lymph Node Metastasis in Human Breast Cancer. Cancer Letters, 253, 34-42.
    http://dx.doi.org/10.1016/j.canlet.2007.01.005

  38. 38. Wang, X.B., Yang, Q.X. and Pei, X.J. (2006) Expression of Angiogenesis-Related Factors in Invasive Breast Cancer and Its Clinical Significance. Journal of Southern Medical University, 26, 860-863.

  39. 39. Hu, S.E., Zhang, Y.J., Cui, Y.M. and Zhang, H.Q. (2005) Expression of VEGF-A and -C in Human Breast Cancer and Their Significance. Chinese Journal of Cancer, 24, 1076-1085.

  40. 40. Yi, W.J., Tang, Z.H., Yang, Z.L., Yu, M.Y., Li, Y.S. and Chen, G.N. (2003) Difference in Expression of VEGF, bFGF and Their Receptors between the Young and Postmenapaosal Women with Breast Cancer. Chinese Journal of Oncology, 25, 141-145.

  41. 41. Linardou, H., Kalogeras, K.T., Kronenwett, R., Kouvatseas, G., Wirtz, R.M., Zagouri, F., et al. (2012) The Prognostic and Predictive Value of mRNA Expression of Vascular Endothelial Growth Factor Family Members in Breast Cancer: A Study in Primary Tumors of High-Risk Early Breast Cancer Patients Participating in a Randomized Hellenic Cooperative Oncology Group Trial. Breast Cancer Research, 14, R145.
    http://dx.doi.org/10.1186/bcr3354

  42. 42. Fuckar, D., Dekanic, A. and Stifter, S. (2006) VEGF and Other Common Tumor Markers in Breast Cancer. International Journal of Surgical Pathology, 14, 49-55.
    http://dx.doi.org/10.1177/106689690601400109

  43. 43. Konecny, G.E., Meng, Y.G., Untch, M., Wang, H.J., Bauerfeind, I., Epstein, M., Stieber, P., et al. (2004) Association between HER-2/neu and VEGF Expression Predicts Clinical Outcome in Primary Breast Cancer Patients. Clinical Cancer Research, 10, 1706-1716.
    http://dx.doi.org/10.1158/1078-0432.CCR-0951-3

  44. 44. Yavuz, S., Paydas, S., Disel, U., Zorludemir, S. and Erdogan, S. (2005) VEGF-C Expression in Breast Cancer: Clinical Importance. Advances in Therapy, 22, 368-380.
    http://dx.doi.org/10.1007/BF02850084

  45. 45. Buteau-Lozano, H., Ancelin, M. and Lardeux, B. (2002) VEGF and Other Common Tumor Markers in Breast Cancer. Cancer Research, 62, 4977-4984.

  46. 46. Sarah, P.G., Margaret, J.C., Cheng, H., et al. (2000) The Short Form of the Alternatively Spliced Flt-4 but Not Its Ligand Vascular Endothelial Growth Factor C Is Related to Lymph Node Metastasis in Human Breast Cancers. Clinical Cancer Research, 6, 4278-4286.

  47. 47. Dakocytomation General Instructions for Immunohistochemical Staining: Manual instructions to the Universal Dakocytomation, LSAB, Dako, Denmark, 2004.

List of Abbreviations

LSAB+: Labeled Streptavidin-biotin,

EGFR: epidermal growth factor receptor,

ER: estrogen,

PR: progesterone,

IHC: immunohistochemistry,

DCIS: ductal carcinoma in situ.

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