Prognostic significance of c-KIT in vulvar cancer: bringing this molecular marker from bench to bedside

Vulvar carcinoma is a rare malignant tumor that presents most commonly as a squamous cell carcinoma (90% of all cases). This neoplasm comprises 3–5% [1‐3] of female genital tract malignant tumors, traditionally being considered a disease of elderly women (aged between 65 and 75 years) [4, 5]. However, in recent decades, its incidence has risen in younger women, [6] possibly due to the higher frequency of precursor lesions–vulvar intraepithelial lesions (VINs)–and their association with Human Papillomavirus (HPV) infection [6, 7]. The medical interest in this type of carcinoma was accentuated in the last decade due to the recognition of the increasing incidence of the disease. Nevertheless, in spite of the increasing interest, limited knowledge of molecular alterations in this type of tumor means that translational research is almost absent in this field.

The c-KIT proto-oncogene, also known as SCFR or CD117 is the v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog. Situated at the long arm of chromosome 4 at 4q11.12, this gene is physiologically expressed in melanocytes, germ cells [8, 9] tissue mast cells, mammary epithelial cells and sudoriferous glands and skin basal layers cells [9‐11]. During development and adulthood, c-KIT modulates various processes including survival, proliferation, migration and differentiation of hematopoietic cells, germ cells and cells derived from the neural crest [12, 13].

A member of the Tyrosine Kinase Receptor Family III (β-PDGFR/CSF-1 Family), c-KIT encodes a transmembrane receptor and has a specific ligand called SCF (stem cell factor, also known as c-KIT ligand or mast cell growth factor) [9]. SCF has two alternatively spliced forms: whereas the soluble form immediately stimulates tyrosine kinase activity, the membrane-bound form produces long-term effects, increasing the stability of the receptor and preventing its rapid downregulation [9]. The interaction between SCF and c-KIT receptor activates the tyrosine kinase function which, in turn, induces phosphorylation of various cellular proteins and activation of a number of cascades, among them via RAS, PI3K and PLCγ, as shown on Figure1.

Various types of neoplasms express c-KIT. In GISTs, for example, this expression is very common [14]. Also, angioleiomyolipomas [15], small-cell lung carcinomas, seminomas/dysgerminomas [10], and leukemias [9, 16] frequently express c-KIT. In many tumor types, this receptor is associated with malignancy. In gastrointestinal stromal tumors, myeloid leukemias and mast cell disorders, for example, c-KIT gene gain-of-function mutations result in constitutive tyrosine kinase activity and are considered to play a central role in oncogenesis and sustained tumor growth [17, 18]. Coexpression of c-KIT and its ligand in small-cell lung cancer, for example, appears to result in an autocrine growth loop sustaining tumor cell proliferation [9, 19]. On the other hand, a markedly better outcome has already been demonstrated in tumors that expressed c-KIT compared with those that did not, such as neuroblastomas [9], nasopharyngeal carcinomas [20] and multiple myeloma [21].

Such controversial findings lead us to investigate the role of this receptor in vulvar carcinomas, as the literature is very limited in this respect and, to our knowledge, no study has been published regarding c-KIT evaluation in squamous cell carcinomas of the vulva (VSCC). For this reason, the present study was designed to evaluate whether c-KIT predicts patient’s outcome in VSCCs and, if so, if this marker can be considered as an efficient prognostic factor. We assessed mRNA expression of c-KIT and its protein product in vulvar squamous cell carcinomas and correlated these data with clinicopathological features and with the presence of HPV infection, in order to determine the prognostic importance of this receptor in vulvar cancer.

As vulvar carcinoma is a relatively rare cancer, very little is known about novel biomarkers with prognostic or predictive roles, although initial efforts are being made. Patients and health services must be made aware of the importance of gynecological evaluation and education policies should be strengthened to increase awareness not only about vulvar carcinoma, but also cervical cancer and HPV infection. In our study, we wished to determine the prognostic importance of c-KIT, a novel prognostic and predictive biomarker for other neoplasms, by evaluating its protein and mRNA expression in vulvar squamous cell carcinomas and correlating its positivity with clinicopathological features and HPV infection.

Our results showed a c-KIT protein positivity by IHC in 70.5% of cases, which was associated with a higher recurrence-free and global survival, an absence of associated lesions or lymph node metastasis and HPV infection, suggesting an important role of this receptor in vulvar carcinoma as a good prognosis marker. Furthermore, c-KIT mRNA quantitation revealed higher transcription levels in normal samples than tumor samples. Thus, this receptor may have potential as a good prognostic marker in vulvar carcinomas.

Proto-oncogene c-KIT encoded tyrosine kinase appears to have an important role in carcinogenesis of various tumors, including gastrointestinal stromal tumors (GIST), melanomas, seminomas, glioblastomas, breast cancer and acute myeloid leukemias (AML) [17, 18]. The c-KIT/SCF system seems to be also involved in carcinogenesis of the female genital tract [23]. In most tumors, progressive decrease in c-KIT expression is reported as long as the tumor grows and invades [23, 24], being positive in normal tissues and decreased in tumor cells. Such data corroborates with our study, since significantly higher expression of c-KIT mRNA was observed in normal tissue when compared to tumor microdissected samples.

In this study, high IHC expression of c-KIT receptor was similar to that described by Eroglu et al. (2008) [25] who found a strong diffuse high expression of protein in the cytoplasm and/or membrane of vulvar epithelioid sarcoma cells. The frequent finding of IHC cytoplasmic staining among our cases suggests an internalization of the receptor after its binding to SCF, which may indicate a physiological regulation of this receptor in the cells, the result of a rapid turnover of the protein when no longer located in the membrane [9, 26].

Longer global and disease free survivals were observed in our patients with positive expression of c-KIT. Similar findings have also been shown by other authors in neuroblastomas [9], nasopharyngeal carcinomas [20] and multiple myeloma [21]. These authors reported that the expression of c-KIT and a better prognosis is an “unexpected” finding since, in several tumor types, c-KIT is associated with malignancy [9] as a result of constitutively turned on tyrosine kinase and, in the case of both expression of c-KIT and its ligand SCF, as a result of self-support from an autocrine tumor growth feedback loop [9, 19]. Bataille et al (2008) [21] found a 93% survival in 4 years for patients with c-KIT positive multiple myeloma versus 64% for c-KIT negative. Similarly, our data show overall survival of 72% for c-KIT positive and 50% for c-KIT negative. Also, a correlation between c-KIT positivity and better recurrence-free survival was observed. Both vulvar skin bridge recurrences and primary tumor site recurrences have been shown in the literature as strong predictors for cancer-related death [27] and, therefore, this supports the utility of this marker as a powerful predictor for tumor recurrence.

The fact that c-KIT exerts divergent functions depending on its modulation by environmental factors, its interactions with several different intracellular effectors pathways and alternative splicing of its mRNA, may account for the differences in function found in different tumors [9, 28, 29]. Studies done in vitro and in vivo in melanoma cells have shown that exposure of c-KIT positive cells to SCF triggers apoptosis, which does not happen in c-KIT negative cells or in normal melanocytes. Since SCF is normally produced by keratinocytes and other dermal cells, loss of expression of c-KIT may allow malignant melanoma cells to escape c-KIT/SCF mediated apoptosis, contributing to growth and tumor metastasis [29, 30].

The most important clinical prognostic factor in vulvar cancer is the lymph node status, which is a part of surgicopathological staging by FIGO [31]. In this sense, it is important to note that a grouping strategy regarding lymph node involvement was made to allow a biologically rational insight of the statistical analysis. Our results show that c-KIT positive cases were associated with absence or involvement of only one lymph node (p = 0.0053) when compared to c-KIT negative cases, which were associated with two or more lymph-node metastasis. This grouping strategy was used by Holschneider and Berek [32] and states that patients with no nodes or only one microscopically involved have a comparable prognosis. The association between positive staining for c-KIT and absence of lymph node metastases, absence of associated lesions, and higher survival rates indicates that this is a marker of good prognosis in vulvar cancer. Possibly, in tumors with positive staining for c-KIT, more conservative surgery with less mutilation should be evaluated in order to provide a better psychosocial quality of life of these women. These psychosocial issues are widely described in the literature as one of the most important concerns especially among younger patients, regarding complications of therapy [31]. Fear, distorted body image and depression, are the most frequent symptoms mentioned in the literature, in this regard [31]. Implementing translational research findings including molecular marker evaluation, such as c-KIT, could be helpful in developing a more personalized approach to the treatment of these patients. Thus, c-KIT positivity could be used as a prognostic factor for vulvar carcinoma.

A relationship between positive expression of c-KIT and the presence of HPV was demonstrated in this study, although there is no data in the literature suggesting this. We hypothesize that (1) the locus of c-KIT gene may be a fragile site yet unknown for integration of HPV; or (2) abnormalities in the regulation of this gene may be accelerating the process of carcinogenesis in cooperation with the viral infection. Besides, the positive correlation between c-KIT expression and HPV reinforces the good prognosis of these tumors, as tumors associated with HPV, which is considered an independent predictor of better survival [33].

Regarding treatment and further predictive values of c-KIT, the positive overexpression of this receptor was found to be associated with a better global and disease free survival in patients with vulvar cancer. For this reason, contrary to the situation found in other c-KIT positive tumors, the use of a c-KIT inhibitor such as Gleevec® may be inappropriate in vulvar cancer. However, a better understanding of the molecular regulatory mechanisms of c-KIT may, in the future, play a role in the development of targeted therapies in order to inhibit or modulate the signaling pathways triggered by this receptor.

Vulvar carcinoma is a rare disease, and thus our study was limited in size. We also encountered problems with lack of clinical information, which reduced numbers further. Scarcity of publications also leads us to a more speculative discussion and comparison with other neoplasms.

The association between positive staining for c-KIT and absence of lymph node metastases, absence of associated lesions, and higher survival rates indicates that this is a marker of good prognosis in vulvar cancer. Possibly, in tumors with positive staining for c-KIT, more conservative surgery with less mutilation should be evaluated in order to provide a better psychosocial quality of life of these women [31]. Fear, distorted body image and depression, are the most frequent symptoms mentioned in the literature regarding the psychosocial quality of life of these patients [31] and could be relieved if more personalized medicine through molecular markers evaluation was used. Thus, molecular marker evaluation (c-KIT positivity), could be used as a prognostic factor for vulvar carcinoma, implementing translational research findings into part of a more personalized approach to each individual patient and their therapy.

BMM conceived the study design, participated in its design and in the acquisition of data, analysis and interpretation. AMLR participated in the acquisition of data, analysis and interpretation. ISR participated in the acquisition of data, analysis and interpretation. GB participated in the acquisition of data. FMC participated in the acquisition of data from medical records. MMS participated in the analysis and interpretation of the data. LTDC performed statistical analysis. KCC participated in the RNA extraction, qRT-PCR procedure and analysis. FAS has been involved in drafting the manuscript or revising it critically for important intellectual content. RMR conceived the study, participated in its design and coordination. All authors have given final approval of the version to be published.