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Expression of CD117 in normal and neoplastic breast tissue: the diagnostic significance and correlation with HER-2 neu, estrogen, and progesterone receptors in breast cancer

El-Sayed, Mona; Shafeek, Mona G.

doi: 10.1097/01.XEJ.0000436653.68526.80
ORIGINAL ARTICLES

Background CD117 (C-KIT) is a tyrosine kinase receptor thought to be involved in the process of malignant transformation.

Objectives The aim of this study was to evaluate the role of C-KIT in malignant transformation and to assess its clinical significance in breast cancer.

Materials and methods Immunohistochemical expression of C-KIT was examined in 50 specimens of paraffin-embedded sections of benign breast lesions (N=20) and invasive ductal carcinoma (N=30), containing normal breast epithelium (N=27). The results were statistically correlated with different clinicopathologic parameters, and HER-2 neu, estrogen receptor (ER), and progesterone receptor (PR) expression.

Results There was a significant difference in C-KIT expression between tumor tissues (16.6%), normal breast tissues (100%) and benign breast lesions (75%) (P<0.001). C-KIT expression in invasive ductal carcinoma was significantly associated with negative lymph node metastasis (P=0.04), the adenoid cystic type (P=0.02) and the loss of PR receptors (P=0.009), but did not show significant difference with respect to the tumor grade, the patient’s age, the tumor size, and HER-2/neu or ER expression.

Conclusion The loss of C-KIT protein may be involved at early stages of human breast cancer development. C-KIT expression in breast cancer may be associated with negative lymph node metastasis and loss of progesterone receptors.

Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Correspondence to Mona El-Sayed, MD, Department of Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt Tel: +20 111 763 3145; e-mail: mona_elbagalaty@hotmail.com

Received June 29, 2013

Accepted July 15, 2013

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Introduction

The proto-oncogene C-KIT located on chromosome 4q11–21 encodes a surface membrane tyrosine kinase receptor (Yarden et al., 1987). The C-KIT is activated by its ligand, stem cell factor. Interaction of the C-KIT receptor and its ligand results in the activation of the KIT kinase domain that plays a role in the activation of a variety of signal transduction pathways involved in proliferation, apoptosis, and tumorigenesis (Heinrich et al., 2002).

C-KIT protein expression has been found in a wide variety of malignant tumors including myeloid leukemia, small cell lung cancer, and gastrointestinal stromal tumors, but it is frequently diminished or lost in other tumors, including malignant melanoma (Wang et al., 1989; Lassam and Bickford, 1992; Plummer et al., 1993; Hirota et al., 1998)

Several studies have shown that C-KIT is highly expressed in the normal breast epithelium and variably expressed in benign breast lesions. In primary breast cancer, C-KIT expression has been reported to vary from 1 to 82%, but its biological and clinicopathological significance is unclear (Natali et al., 1992; Chui et al., 1996; Palmu et al., 2002; Tsuura et al., 2002; Ko et al., 2003; Yared et al., 2004; Ulivi et al., 2004; Tsutsui et al., 2006).

The aim of the present study was to investigate the immunohistochemical expression of C-KIT within the spectrum of normal breast epithelium, benign breast lesions, and breast cancer to evaluate the role of this marker in breast carcinogenesis and other clinicopathologic factors, HER-2 neu, estrogen receptor (ER), and progesterone receptor (PR) expression.

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Materials and methods

Case selection

A total of 50 cases of selected breast tissues were chosen from the archive of the Department of Pathology, Faculty of Medicine, Zagazig University, and from some private histopathology laboratories, during the period from January 2005 to December 2010. No patient’s consents were needed as we used archival materials and the patients were not known. Clinical data of the patients were obtained from medical files. Patients’ age ranged from 29 to 62 years, with a mean age of 38.9±16.3 years. Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. Consecutive 4-µm sections were prepared and stained with hematoxylin and eosin. Histopathological examination revealed fibrocystic disease (N=10), fibroadenoma (N=7), intraductal papilloma (N=3), primary invasive ductal carcinoma (IDC) with their axillary lymph nodes (N=30), and normal breast tissues (N=27) adjacent to benign lesions or cancer breast. All cases of IDC were histologically classified according to the WHO criteria (World Health Organization, 1981) into medullary carcinoma (N=3), tubular carcinoma (N=3), mucinous carcinoma (N=2), papillary carcinoma (N=2), cribriform carcinoma (N=2), adenoid cystic carcinoma (N=2), and other NOS types (N=16), in which six of them have in-situ components. Tumors were graded according to the histological grade using modified the Bloom-Richardson classification (Elston and Ellis, 1991) as grade 1 (N=7), grade 2 (N=15), and grade 3 (N=8). Lymph node metastasis was detected in 14 of 30 cases. We also reviewed immunohistochemically stained slides for ER, PR, and HER-2.

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Immunohistochemistry

The immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue sections using standard streptavidin–biotin–peroxidase complex (ABC) methods. In brief, after routine deparaffinization, 4-µm sections mounted on optiplus positive-charged adhesive slides (Biogenix Co., California, USA) were treated with 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. Sections were then washed in PBS (pH 7.4) and Tris-buffer solution (pH 7.6), respectively. For C-KIT, slides were microwaved for 20 min for antigen retrieval, and incubated overnight at 4°C with anti-C-KIT rabbit polyclonal antibody (1/100 dilution; Dako, Carpentaria, CA, USA). Sections were then treated with the avidin–biotin–peroxidase reagent for 30 min (Dako, Kyoto, Japan). Finally, sections were incubated with diaminobenzidine and counter-stained with hematoxylin, and then cleared and mounted. Sections of normal mammary glands were used as positive controls for C-KIT. In negative controls, the primary antibody was replaced by buffer.

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Scoring criteria

The C-KIT expression level was scored as described by Tsuda et al. (2005) as follows: Score 1+, the cytoplasm was discretely and weakly to moderately stained in 10% or more of the constituent carcinoma cells. Score 2+, the cytoplasm was strongly stained with or without membrane staining in 10% or more of the constituent carcinoma cells. Score 0, no staining was observed or staining was observed in less than 10% of the constituent carcinoma cells. Cases with a score of 1+ and 2+ were considered positive.

ER and PR were scored according to the Allred scoring method Allred et al. (1998). HER-2 staining was scored according to the American Society of Clinical Oncology/College of American Pathologists guidelines (Wolff et al., 2007).

Approved from ethical committee.

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Results

C-KIT expression in normal breast epithelium, benign breast lesions, and breast cancer

Strong positive immunohistochemical staining of C-KIT was observed in the cytoplasm and/or on the cell membrane of the epithelium of normal ductal and lobular breast cells (Fig. 1a and b). In benign breast lesions, moderate to strong C-KIT expression was detected in 15 (75%) of the 20 cases. C-KIT expression was lower in the intraductal papilloma (33.3%) compared with that of fibrocystic disease (80%) and fibroadenoma (71.4%) (Fig. 2a and b). In breast cancer, C-KIT expression was detected in only five (16.6%) of 30 cases (Figs 3 and 4). There was a highly significant difference in the C-KIT expression between cancer breast (16.6%), normal breast (100%), and benign breast lesions (75%) (P<0.001) (Table 1).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Table 1

Table 1

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Relationship between C-KIT expression and some clinicopathologic factors in IDC

C-KIT was significantly expressed in adenoid cystic carcinoma (P=0.02), in which the two studied cases (one was cribriform and the other was of the basal-like type) were strongly positive for C-KIT (+2) (Fig. 3a). An increased frequency of C-KIT expression was also seen in medullary breast cancer, in which one (33.3%) of three tumors was moderately positive for C-KIT protein (+1) (Fig. 3b). In the remaining two cases of KIT-positive IDC NOS with in-situ component, strong C-KIT expression (+2) was observed mainly in stromal cells and in the in-situ component, especially those with comedonecrosis and solid growth patterns (Fig. 4).

C-KIT expression increased from low grade (0%) to high-grade (50%) IDC, but this was statistically not significant (P=0.14). All IDC cases that expressed C-KIT were negative for lymph node metastasis. There was a significant association between negative expression of the C-KIT protein and lymph node metastasis (P=0.04). Also, C-KIT expression did not show significant difference with respect to the patient’s age and the tumor size (Table 2).

Table 2

Table 2

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Relationship between C-KIT, HER-2/neu, ER, and PR

In invasive breast cancer, HER-2/neu expression was detected in only four (13.3%) of 30 cases. However, we found that 50% (two of four cases) of the HER-2/neu-positive cases and 11.5% (three of 26 cases) of the HER-2/neu-negative cases coexpressed C-KIT. This was statistically nonsignificant (P=0.11). ER and PR were detected in 16 (53.3%) and 17 (56.6%) of the 30 cases of IDC, respectively. Only one of the ER (6.2%) and none of the PR-positive IDC coexpressed C-KIT, which was significantly decreased in PR-positive IDC (P=0.009) (Table 3).

Table 3

Table 3

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Statistical analysis

Statistical analysis of data for labeling indices was performed using the χ 2-test or Fisher’s exact test when recommended. P value less than 0.05 was considered statistically significant.

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Discussion

In breast cancer, the expression of C-KIT represents a highly controversial subject (Amin et al., 2012). In the present study, we investigated the expression of C-KIT within the spectrum of normal breast epithelium, benign breast lesions, and breast cancer to evaluate its potential role in malignant transformation and other prognostic parameters.

In our study, there was a highly significant difference in C-KIT expression between tumor tissues (16.6%) and normal (100%) and benign breast lesions (75%) (P<0.001). Similar results were reported by others (Natali et al., 1992; Chui et al., 1996; Palmu et al., 2002; Tsuura et al., 2002; Ko et al., 2003; Ulivi et al., 2004; Yared et al., 2004; Tsutsui et al., 2006), who found that the rate of a positive C-KIT expression in the normal breast tissue was 100% and the rate of positive C-KIT expression or its immunohistochemical score (IRS) in benign tumors was always lower than that of normal breast tissue and always higher than that of breast cancer, which varies from 1 to 82%. In contrast, Eroǧlu and Sari (2007) found that in breast cancer tissues, the mean IRS of C-KIT expression was significantly increased compared with those of fibroadenomas. Also, Roussidis et al. (2007) found a higher rate of C-KIT expression in breast cancer than that of normal breast tissue and benign breast tumors. Moreover, in their work on breast cancer, Amin et al. (2012) found C-KIT expression in 28.6% of the cases, whereas Kashiwagi et al. (2013) found C-KIT expression in 16.6% of the cases, which is very similar to our results.

In this study, C-KIT was significantly expressed in adenoid cystic carcinoma (P=0.02), in which the two studied cases were strongly positive for C-KIT. An increased frequency of C-KIT expression was also seen in medullary breast cancer, in which one (33.3%) of three tumors was positive for C-KIT protein. Nearly similar results were obtained by Azoulay et al. (2005) and Mastropasqua et al. (2005), who found that 95 and 100% of breast adenoid cystic carcinomas were positive for C-KIT and concluded that C-KIT immunoreactivity is the single most reliable diagnostic adjunct in differentiating adenoid cystic carcinomas from infiltrating tubular and cribriform carcinomas, especially in small biopsies. In addition, Nalwoga et al. (2008) and Kashiwagi et al. (2013) found that there is a high expression of C-KIT in high-grade basal-like breast carcinoma. An increased frequency of C-KIT expression in medullary breast cancer was also reported by Simon et al. (2004) who detected positive C-KIT expression in 19.1% of medullary breast cancer. However, they also found C-KIT immunoreactivity in papillary carcinomas, a finding that was not observed in our study. In the present study, C-KIT expression was detected in two cases of IDC NOS with in-situ component, in which its expression was detected mainly in the areas of comedonecrosis and solid growth patterns. These results confirm the observation of Tsuda et al. (2005) and Diallo et al. (2006), who found that C-KIT expression is a frequent finding in comedo and solid growth types ductal carcinoma in-situ that are more often poorly differentiated.

In our study, C-KIT immunoreactivity was detected in the stromal cells of high-grade breast carcinoma. Similar results were also reported by Kondi-Pafiti et al. (2010) and Amin et al. (2012). However, Kondi-Pafiti et al. (2010) reported that C-KIT was highly expressed in the stromal cells of high-grade breast carcinoma despite negative C-KIT expression in tumor cells. Tumor stromal cells may contribute to the establishment of a more permissive microenvironment for tumor growth and progression (Joyce and Pollard, 2009). Moreover, Dabiri et al. (2004) also demonstrated that the presence of C-KIT-positive mast cells in the peritumoral stroma correlated with a good prognosis in breast cancers with long-term follow-up.

In our study, we found a significant association between C-KIT expression and negative lymph node metastasis (P=0.04), but did not find a significant difference with respect to the tumor grade, patients’ age, and the tumor size. The association between negative C-KIT expression and lymph node metastasis has been reported previously by Tsutsui et al. (2006) and Amin et al. (2012). However, Chui et al. (1996), Simon et al. (2004), and Ulivi et al. (2004) did not find any significant correlation between C-KIT expression and lymph node metastasis. In contrast, Shams and Shams (2011) and Kashiwagi et al. (2013), in their work on triple-negative breast cancer, found a significant correlation between C-KIT expression and lymph node metastasis. In addition, a significant correlation between positive C-KIT expression and the tumor grade was proved in some studies (Simon et al., 2004; Tsuda et al., 2005; Diallo et al., 2006; Shams and Shams, 2011), but not proved in others (Nalwoga et al., 2008; Amin et al. 2012). The last authors also did not find a positive correlation between C-KIT expression and patients’ age or the tumor size, which was significantly correlated with C-KIT in another study (Kim et al., 2008).

In this study, we found a nonsignificant association between HER-2/neu, ER, and C-KIT expression. However, C-KIT was significantly decreased in PR-positive IDC (P=0.009). Diallo et al. (2006) and Amin et al. (2012) found that C-KIT expression was significantly lower in ER-positive or PR-positive cases. Also, Shams and Shams (2011) found that the frequency of C-KIT immunoreactivity was 75% in triple-negative breast cancer. In addition, Tsuda et al. (2005) found marginal significance between HER-2/neu-overexpressing and C-KIT-overexpressing cases, However, Diallo et al. (2006) found that C-KIT expression was strongly associated with HER-2 positivity, a finding that was not observed in this study. The reason for this difference is still unclear and might be a sign of dedifferentiation with a different meaning than C-KIT expression in the normal breast epithelium or benign breast lesions (Diallo et al., 2006).

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Conclusion

The loss of C-KIT may be involved at early stages of breast cancer development. C-KIT expression in breast cancer may be associated with negative lymph node metastasis and loss of progesterone receptors. Further studies are required to clarify the active mechanism of C-KIT proto-oncogene production in human breast and to investigate the efficiency of C-KIT in the diagnosis of breast adenoid cystic carcinoma and the possible beneficial effects of C-KIT inhibitor therapy in C-KIT-positive breast cancers.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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