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Overexpression of c-KIT (CD117) in triple-negative breast cancer

Shams, Tahany M.a; Shams, Mohamed E.b

doi: 10.1097/01.XEJ.0000406601.42226.2d
ORIGINAL ARTICLES
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Triple-negative breast cancer (TNBC) is characterized by the lack of expression of the estrogen receptor and the progesterone receptor, and the absence of HER2 protein expression, thus there is no targeted therapy for this subtype. In this study, we aimed to evaluate the expression of c-KIT, which is a tyrosine kinase transmembrane receptor and has its target therapy. Hematoxylin-stained and eosin-stained sections of 72 mastectomy specimens from female patients immunohistochemically confirmed as having TNBC were assessed for grade (G2, n=22; G3, n=50) and pathological stage (PT2, n=12; PT3, n=44; PT4, n=16). Immunostaining was performed for c-KIT using microwave antigen retrieval and an avidin–biotin complex method. The frequency of immunoreactivity was 75%; there was a significant correlation with tumor grade, lymph node metastasis, and lympho-vascular space invasion but no significant correlation with patient’s age, pathological stage, overall survival, and tumor recurrence. Hence, the overexpression of c-KIT in this category of tumor suggests that it may prove useful as a therapeutic target in TNBC.

Departments of aPathology

bGeneral Surgery, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

Correspondence to Assistant Professor Tahany Shams, MD, Department of Pathology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt Tel: +2 01 2879 0821; fax: +2 643206604; e-mail: tahanishams@hotmail.com

Received September 2, 2011

Accepted September 15, 2011

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Introduction

About 10–17% of all breast cancers are found to be triple-negative, which means that cancer cells are negative for estrogen and progesterone hormone receptors and HER2 receptors. Therefore, the cancer will not respond to hormonal therapies and to medications that target HER2. In addition, triple-negative breast cancer (TNBC) tends to be more aggressive than other types of breast cancer. It is more likely to spread beyond the breast, more likely to recur after treatment, and tends to be higher grade than other types of breast cancer (Reis-Filho and Tutt, 2008).

The KIT gene belongs to the family of class III receptor protein tyrosine kinases, which also includes the colony-stimulating factor I receptor, platelet-derived growth factor receptor A and B, and colony stimulating factor 1 receptor (c-Fms) related tyrosine kinase 3 (Andre et al., 1992). KIT is located on chromosome 4q12 and has structural similarities to the other platelet-derived growth factor receptor family members (Qiu et al., 1988; Spritz et al., 1994). Class III receptor protein tyrosine kinases have five extracellular immunoglobulin-like domains, a single transmembrane region, an intracellular juxtamembrane domain, and an intracellular kinase domain split by a kinase insert segment (Ullrich and Schlessinger, 1990).

Signaling by c-KIT appears to play a central role in cellular transformation and differentiation. Therefore, aberrant activation of c-KIT leads to the development (Savage and Antman, 2002) and progression of several human malignancies (Went et al., 2004; Sihto et al., 2005). KIT alterations in malignant tumors are of high interest because KIT is one of the targets of the tyrosine kinase inhibitor imatinib mesylate (STI571; Glivec). STI571 has initially been shown to be effective in the treatment of chronic myeloid leukemia, where it targets the BCR-ABL fusion protein (Kantarjian et al., 2002). More recently, significant treatment responses were also seen in patients with advanced KIT-positive gastrointestinal stromal tumors (Miettinen et al., 2001) and dermatofibrosarcoma protuberance (Sawyers, 2002). In the present study, we investigated the expression of c-KIT in TNBC and its relations with clinicopathological factors such as age, tumor grade, stage, lymph node metastasis (LNM), lympho-vascular space invasion (LVSI), ductal carcinoma in-situ component (DCISC), overall survival, and local tumor recurrence.

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Materials and methods

Seventy-two women diagnosed with TNBC between May 2003 and May 2008 were selected; all of them had invasive duct carcinoma. Their routinely processed archival formalin-fixed paraffin-embedded blocks containing the main tumor were collected from the Pathology Department at Suez Canal University Hospital. Patients’ medical records were reviewed, and clinicopathological characteristics and follow-up data were obtained. We reviewed the immunostained slides for estrogen, progesterone, and HER2 receptor to confirm the negativity for these cases (positive estrogen receptor (ER) or progesterone receptor stains were recorded only if immunostaining was seen within the nuclei of the tumor cells, whereas HER2 required membranous staining; for all, the cut-off point of positivity is ≥10% of tumor cells). The patients’ age ranged from 45 to 77 years, with a median age of 53 years. No patients were diagnosed with histological malignancy grade I, 22 patients were diagnosed with grade II, and 50 with grade III. Two sections of 5-µm thickness were cut from each representative paraffin tissue block. One section was stained with hematoxylin and eosin to confirm the diagnosis, type, and grade of the tumor; another section was used for immunostaining. Histological grading of the invasive carcinomas was carried out according to the system of Nottingham Modification of the Bloom–Richardson (Frierson et al., 1995). The TNM staging system was used for the classification of invasive tumors.

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Immunohistochemistry

Technique of immunostaining

Formalin-fixed, paraffin-embedded tissue sections were mounted on superfrost slides, dewaxed with xylene, and gradually rehydrated. Endogenous peroxidase activity was blocked in (0.3 hydrogen peroxidase for 15 min), and then antigen retrieval was achieved using microwave heating (three times of 10 min; 10 mmol/l citrate buffer, pH 6.0). DAKO Target Retrieval Solution S1699, DAKO Corporation, Carpintera, California, USA). IHC was performed with the anti-CD117 antibody from DAKO (A4502). In a comparison of multiple antibodies, A4502 had previously yielded the highest specificity and the least background (Went et al., 2004). A4502 was applied at a dilution of 1 : 300 at room temperature for 2.5 h. The standard avidin–biotin peroxidase complex technique was carried out using the streptavidin–biotin peroxidase complex kit (DAKO). The sections were incubated with secondary antibody for 15 min and then the detection of bound antibody was accomplished using the ABC reagent for 20 min; each step was followed by PBS wash. Finally, 0.1% solution of diaminobenzidine was used for color development and hematoxylin was used for counterstaining. A preabsorption experiment using CD117 peptide stock solution (Neomarkers PP1518; NeoMarkers, Freemont, California, USA) was used as a negative control. Sections from gastrointestinal stromal tumors were used as positive controls.

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Evaluation of immunostaining

The analysis of all tissue sections was carried out without any prior knowledge of the clinical parameters by means of light microscopy. Staining was considered true positive if the reaction product was localized to the cell membrane alone or to the cell membrane and cytoplasm simultaneously; cytoplasmic staining alone proved to be false-positive in all preabsorption control samples (Went et al., 2004). Staining extent was scored as (a) when 10–50% of the cells were stained, and (b) when more than 50% of the cells were stained. Staining intensity was evaluated only in positive cases and scored as (a) for weak staining (faint, light yellow), (b) for moderate staining (brown), and (c) for strong staining (dark brown) (Aydin et al., 2008).

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Statistical analysis

The data were collected, tabulated, and processed on an IBM PC compatible computer using SPSS version 11 (SPSS, Chicago, IL, USA). Fisher’s exact test or χ2-tests were used to analyze the distribution of c-KIT positive cases according to clinicopathological parameters, overall survival, and tumor recurrence. A P-value of <0.05 was considered statistically significant.

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Results

Demographic data

Among the 72 cases of TNBC studied, all patients were women and their median age was 53 years (range 45–77 years). The demographic details are presented in Table 1. Most patients (61.1 and 69.4%) were in pathological stage T2 and Grade III respectively, 80.6% of the patients had LNM.

Table 1

Table 1

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C-KIT expression (Table 2 and Fig. 1)

Fifty four out of the 72 (75%) studied specimens were positive for c-KIT. The majority of the positive tumor cells showed diffuse cytoplasmic staining with membranous accentuation (Fig. 1a). The rest of the specimens had weak positive staining of less than 10% of the tumor cells and were thus considered as negative. Twenty specimens (37%) showed positive staining of 10–50% of the tumor cells. Thirty four specimens (63%) had positive staining of more than 50% of the tumor cells. Strong positivity (score 3) was found in ten cases (Fig. 1b). The DCISC showed strong membranous and cytoplasmic staining in all cases (Fig. 1c).

Table 2

Table 2

Fig. 1

Fig. 1

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Patients’ follow-up: overall survival and tumor recurrence (Table 1)

Follow-up periods ranged from 8 to 56 months (median 41 months). The overall survival and tumor recurrence were expressed as the number of months from diagnosis to the occurrence of disease-related death or local recurrence, respectively. Follow-up information was available for all studied patients; during their follow-up period, 40 (55.6%) patients died of disease and the recurrence was 24 out of 72 (33.3%) and the median time to recurrence was 15.5 months (4.1–18.2) months. The patients alive were 32 out of 72 (44.4%), and the recurrence free was 48 out of 72 (66.7%).

Regarding the relation between c-KIT expression and overall survival, 34 out of 40 (85%) dead patients were c-KIT positive compared with 20 out of 32 (62.5%) of the surviving patients; this difference in life span was statistically insignificant (P=0.06). In addition, c-KIT-positive staining was detected in 32 out of 48 (66.7%) patients who had been recurrence free during their follow-up compared with 22 out of 24 (91.7%) patients with recurrence; this difference was statistically insignificant (P=0.174).

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Relation between c-KIT expression and clinicopathological characteristics

We then analyzed the relationship between c-KIT expression and the clinicopathological parameters of TNBC. Increased expression was more frequent in TNBC with positive LNM (89.7%) than in negative cases (14.3%), showing a statistically significant relationship (P<0.0001). As shown in Table 1, 22 out of 25 grade III tumors (88%) had positive c-KIT expression, whereas 10 out of 22 (45.5s%) grade II tumors were positive; the relationship between c-KIT expression and tumor grade was statistically significant (P<0.0001). With regard to c-KIT expression in cases with LVSI, there were statistically significant differences between c-KIT-positive and c-KIT-negative cases (P<0.0001). However, no significant differences were found between c-KIT immunoreactivity, age, and DCISC as well as tumor stage (P=0.7, 0.4, and 0.48, respectively) (Table 1).

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Discussion

Breast cancer patients fall into three main groups: (a) those with hormone receptor-positive tumors who are managed with a number of ER-targeted therapy options±chemotherapy; (b) those with HER2+ tumors, who will, in addition, receive HER2-directed therapy; and (c) those with hormone receptor (ER) and progesterone receptor-negative and HER2- negative breast cancers, for whom chemotherapy is the only modality of systemic therapy available. On the basis of the above, it is clear that the interest in triple-negative cancers stems from the lack of tailored therapies for this group of breast cancer patients (Reis-Filho and Tutt, 2008).

Also, triple-negative tumors more frequently affect younger patients (<50 years) (Haffty et al., 2006; Bauer et al., 2007; Dent et al., 2007; Morris et al., 2007; Tischkowitz et al., 2007); as shown in our results, 58.3% of our patients were below 53 years of age and they also had significantly more aggressive tumors compare with the other molecular subgroups (Harris et al., 2006; Bauer et al., 2007; Tischkowitz et al., 2007). This aggressiveness is best illustrated by the fact that the peak risk of recurrence is between the first and the third years and the majority of deaths occur in the first 5 years, following therapy (Dent et al., 2007; Tischkowitz et al., 2007). This is consistent with our results that 55.6 and 33.3% of our studied patients died and were positive for recurrence, respectively.

From a pathologist’s point of view, the differences between triple-negative and non-TNBC are not surprising, given that the majority of triple-negative cancers are of histological grade III (Dent et al., 2007; Rakha et al., 2007); there was a higher prevalence of LNM in triple-negative cancers compared with controls (Dent et al., 2007), which is in agreement with our results, wherein 69.4 and 80.65% of the studied cases were high grade and positive for nodal metastasis, respectively.

Although triple-negative cancers are reported to respond to neoadjuvant chemotherapy (Carey et al., 2007), the survival of patients with such tumors is still poor and their management may therefore require a more aggressive alternative intervention. Thus, the development of biologically informed systemic therapies and targeted therapies for triple-negative cancers is of paramount importance.

The c-KIT proto-oncogene codes for a type III tyrosine kinase receptor protein (CD117) that is structurally related to other transmembrane receptors, including platelet-derived growth factor and colony-stimulating factor 1 (Besmer et al., 1986; Qiu et al., 1988). There is some variability in the KIT positivity rates reported in breast cancer (1–13%; Natali et al., 1992; Chui et al., 1996; Tsuura et al., 2002; Simon et al., 2004; Went et al., 2004), and c-KIT has been shown to be preferentially expressed in basal-like subtype invasive breast carcinoma (Nielsen et al., 2004).

In this study, we used the antibody A4502 (DAKO) as it was found to be optimal in staining property, because it had the highest frequency of positivity in arrayed gastrointestinal stromal tumors and because reagents are available for preabsorption control experiments to ensure maximum specificity of the system (Simon et al., 2004).

To our knowledge, at the time of drafting this paper, no immunohistochemical analysis of c-KIT in TNBC has been reported. According to the current study, c-KIT was detected in 75% of the studied cases. Patients with c-KIT-positive tumors were younger than the c-KIT-negative patients but the difference is not statistically significant. Our study revealed a significant association between tumor grade and c-KIT positivity (88.0% at GIII vs. 45.5% at GII, P<0.0001); we also reported a statistically significant relation between c-KIT expression and LNM and LVSI, with a P-value less than <0.0001 for both. The possible association between the expression of c-KIT and DCISC and tumor stage remains unclear; no significant correlation was demonstrated in this study.

The role of c-KIT expression and prognosis was evaluated in other tumors rather than TNBC; one of these studies showed a significant association with poor survival (Micke et al., 2003) and others revealed a significant association (Potti et al., 2003; Pan et al., 2005). In the current study, although a trend toward poor survival and high local tumor recurrence was seen for c-KIT positive patients, it did not reach statistical significance; it had not been clearly defined and needs further studies in large series.

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Conclusion

Triple-negative cancer constitutes one of the most challenging groups of breast cancer; it is associated with a high expression of c-KIT immunohistochemically, high grade, high recurrence rate, and increased LNM. It has been hypothesized that imatinib mesylate could be used as a tailored therapy for triple-negative cancers, on the basis of its inhibition of c-KIT. We recommend further studies to determine whether c-KIT expression is correlated with c-KIT DNA mutations and to test the possibility of treatment with imatinib mesylate (Gleevec).

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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