| Abstract|| |
Background and Objective: In breast cancer, the expression of CD117 represents a highly controversial subject but the majority of studies have found decreased c-kit expression in malignant breast epithelium. A number of studies have reported that increased intratumoral microvessel density (MVD) is associated with poor prognosis in breast cancer. The aim of the study was to assess the relation of CD117 and MVD with other clinicopathological parameters in invasive breast carcinomas using the tissue microarray technique. Materials and Methods: A total of 126 cases of invasive breast carcinoma of different histological types and grades were collected from files of a pathology department during 2010. Clinicopathological and histological parameters were evaluated. Sections from formalin-fixed, paraffin-embedded tumor tissues microarray blocks were immunostained with CD117 and CD34. Statistical analysis of data was done using SPSS, version 16.0. Results: About 29% of invasive breast carcinomas were CD117 positive. There were significant differences between expression of CD117 in the tumor epithelial cells and age of the patient; tumor grade; tumor size, and LN metastasis. Also, there was significant relation between expression of CD117 in the tumor epithelial cells and MVD, expression of estrogen, and progesterone receptors. On multivariate analysis, the most important predictors of negativity of CD117 were tumor size and positive lymph node involvement. Conclusion: Lack of CD117 immunoreactivity in invasive breast carcinoma was associated with features of more aggressive tumor behavior as higher microvessel density, larger size, higher tumor grade, more lymph node metastasis, and negative estrogen and progesterone receptors.
Keywords: Breast carcinoma, CD117, microvessel density
|How to cite this article:|
Amin MM, El-Hawary AK, Farouk O. Relation of CD117 immunoreactivity and microvascular density in invasive breast carcinoma. Indian J Pathol Microbiol 2012;55:456-60
|How to cite this URL:|
Amin MM, El-Hawary AK, Farouk O. Relation of CD117 immunoreactivity and microvascular density in invasive breast carcinoma. Indian J Pathol Microbiol [serial online] 2012 [cited 2017 Jul 21];55:456-60. Available from: http://www.ijpmonline.org/text.asp?2012/55/4/456/107780
| Introduction|| |
The proto-oncogene c-kit (CD117) is a transmembrane tyrosine kinase growth factor receptor  that maps to chromosome 4 (q11-12) and is related structurally to the platelet-derived growth factor/colony stimulating factor-1 receptor family of proteins.  Dimerization and autophosphorylation of CD117, upon binding to its specific ligand, known as stem-cell factor, [3 ] is known to inhibit apoptosis and potentiate cell proliferation. , CD117 plays an important role in the development of gastrointestinal stromal tumors, small-cell lung cancer, melanoma, and breast cancer  but is frequently diminished or lost in other tumors, including malignant melanoma. 
Several studies have shown that c-kit is highly expressed in normal breast epithelium, but is present at only low levels or is completely lost in primary invasive breast cancer or breast cancer metastases. , Since there is a reduction of c-kit expression in malignant transformation in breast epithelium, it is believed that it has a carcinogenesis role in the breast.  In recent years, the role of c-kit has been studied in specific subtypes of breast carcinomas, associated with different clinical outcomes. C-kit appeared to be an indicator of high-grade breast carcinoma groups that often contain the carcinomas with mesenchymal and/or myoepithelial differentiation (basal-like subtype), which have a poor prognosis. 
The formation of new microvessels from preexisting vessels is necessary for tumor growth, and subsequent tumor metastasis.  A number of studies have reported that a hematogenous spread of tumor cells is quantitatively related to intratumoral microvessel density (MVD). These data show that increased MVD is associated with a higher incidence of metastasis and a poor prognosis in various malignancies, including breast cancer.  The aim of this study was to assess the relation of CD117 and MVD with other clinicopathological parameters in invasive breast carcinomas using the tissue microarray technique.
| Materials and Methods|| |
A total of 126 cases of invasive breast carcinoma of different histological types and grades were collected from files of a pathology department. Specimens were obtained from patients who underwent modified radical mastectomy during 2010. This study was approved by the research ethics committee of the university. Clinicopathological parameters such as age, histopathological type, histological grade, tumor size, and lymph node metastasis were evaluated. Histological typing of breast carcinoma cases were done according to the WHO criteria.  The histological grade was assessed using modified Bloom-Richardson classification.  Histological features including, tumor margin, presence or absence of necrosis, associated ductal carcinoma in situ, and lymphovascular invasion were evaluated. We also reviewed immunohistochemically stained slides for ER, PR, and HER2. We constructed tissue microarray blocks using Shebl et al.'s technique.  With mechanical pencil tip (Rotring Tikky II Mechanical Pencil 1.0 mm), a hole (diameter = 1 mm) was punched into a recipient paraffin block and then a cylindrical core sample (diameter = 1 mm), which had been punched from the donor tissue block, was deposited into the hole. Sections from formalin-fixed, paraffin-embedded tumor tissue array blocks were immunostained with CD117 (polyclonal rabbit antihuman code A4502; dilution 1:50; Dako Cytomation, Denmark) and CD34 (Monoclonal mouse antihuman code IR632, clone QBEnd 10, prediluted; Dako Cytomation, Denmark) using standard immunoperoxidase methods. Primary antibodies were left to react for 30 min at room temperature. The negative control was done by omission of primary antibody. Examination of three tumor slides from each specimen was done on an Olympus CX31 light microscope. Pictures were obtained by a PC-driven digital camera (Olympus E-620). The computer software (Cell*, Olympus Soft Imaging Solution GmbH) allowed morphometric analysis to be performed.
ER and PR were scored according to the Allred scoring method.  HER2 staining was scored according to the American Society of Clinical Oncology/College of American Pathologists guidelines. 
Assessment of CD117
Both membranous and cytoplasmic staining of CD117 were evaluated in tumor epithelial and stromal cells. We used the proposed scoring system from studies of Tsuda et al.  and Diallo et al.:  Score 0, no staining was observed or staining was observed in less than 10% of epithelial cells; score 1+, the cytoplasm was discretely and weakly moderately stained in 10% or more of the epithelial cells; score 2+, the cytoplasm was strongly stained with or without membrane staining in 10% or more of the epithelial cells. Cases with score 0 are considered negative and cases with a score of 1+ and 2+ were considered positive.
Assessment of CD34
The number of vessels was scored according to Lu et al.  In high power microscopic views, all the positive microvessels were counted for MVD. Any single positively colored cell or group of cells was evaluated. Vessels laying beside each other were counted independently if only one stromal cell was found between them.
The median value of MVD of the means of all cases (which was 19 in our study) was used as the respective cut-off point and tumors were classified as either less than or greater than the median value.
Data were analyzed applying SPSS, using version 16.0. Variables were presented as number and percent. χ2 -test was used to test significance between groups. A significant predictor in bivariant analysis was entered into the logistic regression model. Odds ratios and their confidence intervals were calculated. P value ≤ 0.05 was considered significant.
| Results|| |
In total, 126 informative cases were analyzed. Ages ranged from 23 to 65 years (mean, 42.9 years), and all were women. The histological types were invasive ductal carcinoma (IDC) (96 cases, 76.2%); invasive lobular carcinoma (ILC) (10 cases, 7.9%); mixed IDC and ILC (8 cases, 6.3%); mucinous carcinoma (6 cases, 4.8%); medullary carcinoma (4 cases, 3.2%), and tubular carcinoma (2 cases, 1.6%). The tumors were classified as grade I (12.7%), grade II (61.9%), and grade III (25.4%). Tumor sizes varied from 1.5 to 12 cm, with a mean size of 5.7 cm. At the time of mastectomy, 100 patients (80.2%) displayed positive lymph nodes [Table 1].
|Table 1: Relationship between expression of CD117 in tumor cells and clinicopathological parameters|
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Thirty-six cases (28.6%) of invasive breast carcinoma were positive for CD117 in tumor epithelial cells including 24 cases of IDC [Figure 1]a and b, all cases of ILC [Figure 1]c, and two cases of mixed ductal and lobular invasive carcinomas [Figure 1]d. There were statistically significant differences between expression of CD117 in the tumor epithelial cells and age of the patient (P = 0.02); tumor grade (P = 0.02); tumor size (P = 0.000); and LN involvement (P = 0.000) [Table 1]. Stromal cells were only focally observed grade I and II carcinomas (both ductal and lobular), and the c-kit expression in these types of cells was very difficult to reveal. In contrast, CD117-positive stromal cells were remarkably numerous in grade III carcinomas [Figure 1]b. Nearly equal numbers of invasive breast carcinoma showed MVD less than the median value [Figure 2]a and greater than the median value [Figure 2]b.
|Figure 1: CD117 immunostaining. (a) GIII infiltrating duct carcinoma shows strong cytoplasmic expression in >10% tumor cells (score + 2). (b) GIII infiltrating duct carcinoma shows strong expression of tumor and stromal cells (score + 2). (c) ILC shows strong cytoplasmic expression in >10% of tumor cells (score + 2). (d) Mixed IDC and ILC show moderate cytoplasmic expression >10% of tumor cells (score + 1) (immunoperoxidase a-d, ×200)|
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|Figure 2: CD34 immunostaining with only blood vessels endothelium is positive. (a) CD34 positive cells less than the median value. (b) CD34 positive cells greater than the median value (MVD) (immunoperoxidase A, ×100; B, ×200)|
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There was significant difference between expression of CD117 in the tumor epithelial cells and MVD (P = 0.002); expression of estrogen receptors (P = 0.009) and progesterone receptors (P = 0.01). No significant difference was found between expression of CD117 in tumor cells and tumor margin (P = 0.053); necrosis (P = 0.15); the presence or absence of ductal carcinoma in situ (P = 0.35); lymphovascular invasion (P = 0.35), and Her2-neu receptors expression (P = 0.54) [Table 2].
|Table 2: Relationship between expression of CD117 in tumor cells and histological features including MVD|
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On multivariate analysis, the most important predictors of negativity of CD117 in this study were the tumor size (P = 0.005) and positive lymph node involvement (P = 0.001). Cases of tumor size 5 cm or more are four times (95% CI: 1.5-8.4) more likely to be negative for CD117. Cases with positive lymph node involvement are five times (95% CI: 1.9-13.6) more likely to be CD117 negative.
| Discussion|| |
The expression of c-kit has been reported in normal breast epithelium and in neoplastic cells.  In breast cancer, the expression of c-kit represents a highly controversial subject, but the majority of studies have found decreased c-kit expression in malignant breast epithelium.  In this study, we reported prevalence of CD117 immunoreactivity in invasive breast carcinoma (28.6%), a figure which was slightly lower than previously published data documenting immunoreactivity in 36% of the cases.  Lennartsson et al.  described the loss of c-kit expression during the progression of normal tissue to breast cancer. Proliferation and/or the differentiation of normal breast cells must be regulated through the c-kit signaling pathway.  Reduction of c-kit expression in malignant transformation in breast epithelium may suggest its carcinogenic role in the breast cancer  as the transformed cells tend to escape the regulatory mechanisms.  A high level of c-kit expression is infrequent in breast cancer.  Eroğlu and Sari  reported that a high level of c-kit expression occurs frequently in invasive breast cancer.
In our study, lack of CD117 immunoreactivity was significantly related to the indicators of aggressiveness in breast cancer, such as the higher tumor grade, larger size, and more lymph node metastasis. Tsutsui et al.  reported that loss of c-kit positivity is usually associated with more advanced breast cancer. However, Eroğlu and Sari  found no statistically significant relationship between the expression of c-kit proto-oncogene product in breast cancer tissue and histological type, tumor size, lymph node metastasis, distant metastasis, stage, and age of the patients.
A number of studies have reported that a hematogenous spread of tumor cells is quantitatively related to intratumoral MVD. We found that higher MVD was more prevalent in negative CD117 invasive breast carcinoma. These findings may support a possible new role for c-kit in tumor progression through its interaction with stromal elements. It might be expected that microvessel density would be augmented in CD117+ carcinomas as Pelosi et al.  found that CD117 immunoreactivity was more prevalent in lung squamous cell carcinoma with greater microvessel density. However, such a fact was not observed in our study.
Kondi-Pafiti et al.  reported that CD117 was highly expressed in stromal cells of high grade breast carcinoma despite negative c-kit expression in tumor cells. Also, in our study these types of cells were remarkably numerous in the stroma of high-grade carcinomas. Stromal cells are well known to play a pivotal role in promoting tumor growth as tumor stromal cells may contribute to the establishment of a more permissive microenvironment for tumor growth and progression.  Dabiri et al. also demonstrated that the presence of c-kit positive mast cells in peritumoral stroma correlated with a good prognosis in breast cancers with long-term follow-up
There was statistically significant difference between expression of CD117 in the tumor epithelial cells and expression of estrogen receptors and progesterone receptors. This in concordance of the results of Diallo et al.,  who reported that c-kit expression was significantly lower in ER- or PR-positive cases. Also, they found that c-kit expression was strongly associated with HER-2 positivity. They suggested that the implications of c-kit and Her2-neu coexpression for breast carcinogenesis should be evaluated. However, we could not elucidate this fact in our study.
In this study, the most important predictors of negativity of CD117 were the tumor size and positive lymph node involvement. Pelosi et al.  confirmed that the increase of tumor diameter was independently associated with CD117 expression in tumor cell of lung adenocarcinoma. Since breast cancer is typically described in stages according to the size of the tumor, nodal status and the presence of distant metastasis,  evaluation of CD117 expression may be helpful prognostic indicator, as loss of c-kit positivity is usually associated with more advanced stage of breast cancer.
| Conclusion|| |
Lack of CD117 immunoreactivity in invasive breast carcinoma was associated with features of more aggressive tumor behavior as higher microvessel density, larger size, higher tumor grade, more lymph node metastasis, and negative estrogen, and progesterone receptors.
| Acknowledgments|| |
The authors thank Prof. Abdel Hady Al-Gilany, Department of Public Health and Preventive Medicine, Mansoura University, for his valuable advices regarding the statistical analysis of the data included in this article.
| References|| |
|1.||Morini M, Bettini G, Preziosi R, Mandrioli L. C-kit gene product (CD117) immunoreactivity in canine and feline paraffin sections. J Histochem Cytochem 2004;52:705-8. |
|2.||Yarden Y, Kuang WS, Yang-Feng T, Coussens L, Munemitsu S, Dull TJ, et al. Human protooncogene c-kit: A new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J 1987;6:3341-51. |
|3.||Cetin N, Dienel G, Gokden M. CD117 expression in glial tumors. J Neurooncol 2005;75:195-202. |
|4.||Kitamura Y, Hirotab S. Kit as a human oncogenic tyrosine kinase. Cell Mol Life Sci 2004;61:2924-31. |
|5.||Chui X, Egami H, Yamashita J, Kurizaki T, Ohmachi H, Yamamoto S, et al. Immunohistochemical expression of the c-kit proto-oncogene product in human malignant and non-malignant breast tissues. Br J Cancer1996;73:1233-6. |
|6.||Tsuura Y, Hiraki H, Watanabe K, Igarashi S, Shimamura K, Fukuda T, et al. Preferential localization of c-kit product in tissue mast cells, basal cells of skin, epithelial cells of breast, small cell lung carcinoma and seminoma/dysgerminoma in human: Immunohistochemical study on formalin-fixed, paraffin-embedded tissues. Virchows Arch 1994;424:135-41. |
|7.||Lassam N, Bickford S. Loss of c-KIT expression in cultured melanoma cells. Oncogene 1992;7:51-6. |
|8.||Ko CD, Kim JS, Ko BG, Son BH, Kang HJ, Yoon HS, et al. The meaning of the c-kit protooncogene product in malignant transformation in human mammary epithelium. Clin Exp Metastasis 2003;20:593-7. |
|9.||Tsuura Y, Suzuki T, Honma K, Sano M. Expression of c-KIT protein in proliferative lesions of human breast. Sexual difference and close association with phosphotyrosine status. J Cancer Res Clin Oncol 2002;128:239-46. |
|10.||Roussidis AE, Theocharis AD, Tzanakakis GN, Karamanos NK. The importance of c-KIT and PDGF receptors as potential targets for molecular therapy in breast cancer. Curr Med Chem 2007;14:735-43. |
|11.||Tsuda H, Morita D, Kimura M, Shinto E, Ohtsuka Y, Mat- subara O, et al. Correlation of KIT and EGFR overexpression with invasive ductal breast carcinoma of the solid-tubular subtype, nuclear grade 3 and mesenchymal of myoepithelial differentiation. Cancer Sci2005;96:48-53. |
|12.||Baeriswyl V, Christofori G. The angiogenic switch in carcinogenesis. Semin Cancer Biol 2009;19:329-37. |
|13.||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. |
|14.||EllisI O, Schnitt SJ, SastreGarau X, Bussolati G, Tavassoli FA, Eusebi V, et al. Invasive breast carcinoma. In: Tavassoli FA, Devilee P, editors. World Health Organization classification of tumors. Pathology and genetics of tumors of the breast and female genital organs. Lyon: IARC Press; 2003. p. 13-59. |
|15.||Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. The value of histological grade in breast cancer: Experience from a large study with long-term follow-up. Histopathology 1991;19:403-10. |
|16.||Shebl AM, Zalata KR, Amin MM, El-Hawary AK. An inexpensive method of small paraffin tissue microarrays using mechanical pencil tips. Diagn Pathol 2011;6:117. |
|17.||Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998;11:155-68. |
|18.||Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 2007;25:118-45. |
|19.||Diallo R, Rody A, Jackish C, Ting E, Schaefer KL, Kissler S, et al. C-kit expression in ductal carcinoma in situ of the breast: Co-expression with HER-2/neu. Hum Pathol 2006;37:205-11. |
|20.||Lu J, Wang J, Wang T, Wang Y, Wu W, Gao L. Microvessel density of malignant and benign hepatic lesions and MRI evaluation. World J Gastroenterol 2004;10:1730-4. |
|21.||Crisi GM, Marconi SA, Makari-Judson G, Goulart RA. Expression of c-kit in adenoid cystic carcinoma of the breast. Am J Clin Pathol 2005;124:733-9. |
|22.||Kondi-Pafiti A, Arkadopoulos N, Gennatas C, Michalaki V, Frangou-Plegmenou M, Chatzipantelis P. Expression of c-kit in common benign and malignant breast lesions. Tumori 2010;96:978-84. |
|23.||Lennartsson J, Voytyuk O, Heiss E, Sundberg C, Sun J, Rönnstrand L. C-KIT signal transduction and involvement in cancer. Cancer Ther 2005;3:5-28. |
|24.||Mundim FG, Logullo AF. Role of the expression of the tyrosine kinase receptor KIT in invasive ductal carcinomas of the breast: A review. Appl Cancer Res 2009;29:50-7. |
|25.||Ko CD, Son BH, Ahn SH, Ka IW, Gong GY, Kim JC. Significance of mismatch repair protein expression in the chemotherapeutic response of sporadic invasive ductal carcinoma of the breast. Breast J 2004;10:20-6. |
|26.||Simon R, Panussis S, Maurer R, Spichtin H, Glatz K, Tapia C. KIT (CD117)-positive breast cancers are infrequent and lack KIT gene mutations. Clin Cancer Res 2004;10:178-83. |
|27.||Eroðlu A, Sari A. Expression of c-kit proto-oncogene product in breast cancer tissues. Med Oncol 2007;24:169-74. |
|28.||sutsui S, Yasuda K, Suzuki K, Takeuchi H, Nishizaki T, Higashi H, et al. A loss of c-KIT expression is associated with an advanced stage and poor prognosis in breast cancer. Br J Cancer 2006;94:1874-8. |
|29.||Pelosi G, Barisella M, Pasini F, Leon ME, Veronesi G, Spaggiari L, et al. CD117 immunoreactivity in stage I adenocarcinoma and squamous cell carcinoma of the lung: Relevance to prognosis in a subset of adenocarcinoma patients. Mod Pathol 2004;17:711-21. |
|30.||Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer 2009;9:239-52. |
|31.||Dabiri S, Huntsman D, Makretsov N, Cheang M, Gilks B, Bajdik C, et al. The presence of stromal mast cells identifies a subset of invasive breast cancers with a favorable prognosis. Mod Pathol 2004;17:690-5. |
|32.||American Joint Committee on Cancer.: AJCC Cancer Staging Manual. 6 th ed. New York, NY: Springer -Verlag, 2002, pp 227-8 |
Amira K El-Hawary
Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]