Serum Survivin in Oral Submucosal Fibrosis and Squamous Cell Carcinoma

Abstract

Survivin, an inhibitor of apoptosis protein is a biomarker of significance in prognostication of many malignancies. In the current study we investigated the serum survivin levels in patients with oral submucosal fibrosis (OSMF) and squamous cell carcinoma (OSCC). Serum was isolated from, peripheral blood collected of clinically and histopathologically confirmed OSMF and OSCC patients. Circulating level of survivin was measured in patients and control subjects by ELISA and analyzed further using Kruskal–Wallis test and two-sample Wilcoxon rank-sum (Mann–Whitney) test. Serum Survivin levels were significantly reduced in the OSCC group as compared to the control group. No significant correlation was noted between the serum survivin level and various clinicopathological characteristics of OSCC and OSMF patients. Our study suggests that free, wild form of circulating survivin probably has no role in predicting the prognosis of oral cancer or the malignant transformation potential of oral submucosal fibrosis.

Introduction

Malignancy of oral cavity is one among the three most common types of cancer in India [1] and oral squamous cell carcinoma accounts (OSCC) for 90% of these oral cancers in south central Asia [2]. Oral squamous cell carcinoma, is the sixth most common malignancy in the world [3] has a poor prognosis despite the recent advances in management. The prognosis of the malignancy worsens with the advancing stage of the disease. Some important factors contributing to the poor prognosis include the inability to address the tumor at a premalignant stage, difficulty in addressing the tumor at an advanced stage due to its malignant behavior, high risk of locoregional recurrence and a lack of proper prognostication and target molecular markers that are unique and significant for oral squamous cell carcinoma.

Majority of the OSCC arise from premalignant lesions like oral submucosal fibrosis (OSMF) and leukoplakia [4]. OSMF is a potential premalignant condition of the oral cavity with around 7.6% malignant transformation rate [5]. The disease has a regional distribution and is particularly common in countries where betel quid and guthka (a form of chewing tobacco) is frequently used such as India and other parts of Asia. However, it is also prevalent among the Asian populations of USA, UK and other developed countries [6] and thus has become a global issue. Therefore, a specific biomarker for early detection of OSCC and/or at a potential premalignant stage is highly required for better diagnosis, prognosis and treatment.

Survivin is an Inhibitor of Apoptosis Protein (IAP) that has recently gained popularity as a tumor marker for many malignancies. It has been found to be differentially expressed in most of the malignancies as compared to the other members of its family. The various molecular profiling studies and retrospective analyses showed increased expression of survivin as risk factor for cancer progression and poor prognosis [7]. The action of survivin bisects various pathways of tumorigenesis like cell proliferation [8], apoptosis inhibition [9], angiogenesis [10] and metastasis [11]. Among these, the major functions identified to date are antiapoptotic property and role in cell cycle control [12]. Survivin is also essential for the hematopoietic progenitor cells proliferation and is also expressed in the proliferating normal adult tissues like neurons and hepatocytes. However, the complete significance of this is yet to be defined [7].

The role survivin in pathogenesis of the premalignant condition i.e. OSMF or OSCC is not yet completely known. Most of the literature is available survivin expression suggests its correlation with poor prognosis, higher stage of tumor and lymph node metastasis [13]. Immunohistochemistry showed that the survivin expression levels were highest in the OSCC group as compared to the OSMF and control groups. The difference in the survivin expression was also reported for the different stages of OSMF [14]. The presence of survivin has not only been identified in various subcellular sites but also in the extracellular space. The exosome mediated release of survivin into the extracellular space has been described basally and after stress [15]. However, the studies on the significance of serum level of survivin in predicting malignant transformation of OSMF and aggressiveness of oral squamous cell carcinoma has not been investigated so far. The present study was conducted to compare the peripheral circulating levels of serum survivin in OSMF and OSCC patients and compare it with healthy controls.

Methods

Patients and Controls

Sixty biopsy proven patients, 30 with OSMF and 30 with OSCC attending the out and in patient at department of Otolaryngology at All India Institute of Medical Sciences (AIIMS), New Delhi were enrolled in the study. OSCC was staged according to the American Joint Committee on Cancer staging [16]. Clinical and histopathological staging of OSMF patients was done as per criteria laid down by More, and Pindborg and Srisat respectively [17]. The control group included 30 age matched healthy subjects. The study was approved by the ‘Ethics Committee’ of All India Institute of Medical Sciences and informed consent was obtained from all participants. Clinicopathological features of study subjects is shown in Table 1.

Table 1.

Clinicopathological features of patients and controls

Characteristics	OSCC patients	OSMF patients	Controls
Age [year]	28–60	21–60	23–55
Mean	45.2	35.6	32.8
Gender
Male	27 [90%]	24 [80%]	20 [66.66%]
Female	3 [10%]	6 [20%]	10 [33.33%]
Site of the tumor
Tongue	14 [46.6%]
Buccal mucosa	7 [23.3%]
Gingivobuccalsulcus	5 [16.66%]
RMT	2 [6.66%]
Alveolus	1 [3.33%]
Lip	1 [3.33%]
Stage of the tumor
T1	2 [6.66%]
T2	4 [13.33%]
T3	6 [20%]
T4	18 [60%]
Lymph node metastasis
N0	10 [33.33%]
N1	9 [30%]
N2	9 [30%]
N3	2 [6.66%]
Histological stage
WDSCC	6 [20%]
MDSCC	7 [23.3%]
PDSCC	17 [56.66%]
OSMF clinical stage
S2		3 [10%]
S3		22 [73.33%]
S4		5 [16.66%]
Mouth opening
M1		16 [53.33%]
M2		14 [46.66%]
Histological grading
Very early		6 [20%]
Early		7 [23.3%]
Moderately advanced		14 [46.66%]
Advanced		3 [10%]

Serum Preparation and Storage

About 5.0 ml of venous blood sample was withdrawn from the patients and controls under aseptic precautions. The sample was collected in a syringe without any anticoagulant and transferred to a micro centrifuge tube. It was then centrifuged at 2000 rpm for 20 min. The supernatant was taken into vials of 100 μl and stored at − 80 °C till further use.

ELISA

The circulating level of survivin in the patient and control subjects was determined by using a commercially available ELISA kit (YH biosearch laboratory, Shanghai, China) as per the manufacturer’s instructions. All the serum samples were estimated in duplicates. The samples optical density (OD) was measured at 450 nm immediately after the experiment and concentrations of survivin in the respective samples were determined using the standard curve.

Statistical Analysis

The statistical analysis of data was performed using software Stata version 14.0. The difference between the experimental and control group variables was calculated using Mann–Whitney and Kruskal–Wallis test. The p value < 0.05 was considered significant.

Results

Clinicopathological Profile of OSCC and OSMF

The age and gender of the patients and controls as well as clinicopathological features of tumors have been shown in Table 1.

In OSCC group majority [80%] of the patients had presented with late stage [III and IV] and the rest [20%] had early stage [I and II] tumors. In OSCC, tongue [46.6%] was the most commonly affected sub site followed by buccal mucosa [23.3%]. Histopathological analysis revealed moderately differentiated carcinomas patients in most [56.6%] patients as compared to poorly differentiated carcinomas [23.3%] and well differentiated carcinomas [20%] in remaining patients.

In cases with OSMF majority [73.3%] of the patients had clinically stage III disease (Table 1).

Mouth opening was up to or greater than 35 mm in 16 [53.3%] of patients and between 25 and 35 mm in 14 [46.6%] of patients. Histopathological grading showed moderately advanced disease in 14 [46.6%] patients, early disease in 7 [23.3%] patients, very early disease in 6 [20%] and advanced disease in 3 [10%] patients.

Circulating Survivin Levels in Patients and Controls

Serum survivin level in all patients and controls is represented in Fig. 1. The mean survivin level in the OSCC, OSMF and control groups was 596.36, 779.23 and 1094.46 pg/ml respectively. The results showed that the circulating survivin level was significantly higher in the control group than the OSCC group [p < 0.001]. There was no significant difference in the circulating survivin levels between the OSCC and OSMF group. Similarly, no significant difference in the circulating survivin level was noted between the OSMF and control group. Serum circulating level did not show any correlation with clinicopathological characteristics of OSCC as well as OSMF.

Fig. 1.

Dot plot representing serum levels of survivin OSMF, OSCC and control subjects

Discussion

Earlier several studies have been performed to measure the survivin expression in various cancers including oral cancer. Some recent studies have also demonstrated differential expression of not only survivin but also its isoforms in OSCC. In one study the expression of 5 survivin isoforms [wild type (wt), 2B, 3B, 2α, and survivin ΔΕ × 3) by RT PCR showed variable expression of these isoforms in the tumor samples as compared to normal tissues [18]. Another study showed that survivin and its isoforms such as Survivin ΔΕ × 3, Survivin 2B were significantly expressed in OSCC tissue as compared to the normal tissue when tested by RT-PCR and immunohistochemistry (IHC). However, any of the isoforms did not show significant correlation with clinicopathological parameters such as stage, grade and lymphnode metastasis of the tumor [19]. In contrast, decreased survivin expression has been shown in tumor and lymphnode metastasis samples as compared to the noncancerous tissues of the head and neck carcinoma (HNSCC). It was further shown that the patients overexpressing survivin had significantly better overall survival as compared to the patients who did not express it [12]. Although there have been many studies that reported the significance of survivin expression in oral cancer, most of these studies showed survivin expression in the tumor and normal tissues by using IHC and RT-PCR techniques. Unfortunately, no study has shown any standard pattern of survivin expression and/or correlation between the various prognostic factors and the biomarker expression. In addition, there are no reports on the role of survivin in the progression of premalignant lesion to malignant condition. Hence we chose to evaluate the significance of the circulating serum survivin in oral cancer and its role in the malignant transformation of a potentially premalignant condition i.e. oral sub mucosal fibrosis.

In the present study, serum survivin levels were significantly reduced in the OSCC group as compared to control group. No significant correlation was noted between circulating survivin levels and various clinicopathological characteristics of OSCC and OSMF. Although survivin has been highlighted as a tumor marker that is selectively elevated in a number of malignancies, recent research has demonstrated its presence in normal adult tissues and its role in regulating cell proliferation and survival [7]. Almost around ten splice variants of survivin have been identified, and the function of these variants has not yet been defined completely. These splice variants have varying levels of antiapoptotic activity and one of them has shown apoptotic activity as well [20]. The higher levels of splice variants have been encountered in the tumor specimens as compared to the normal tissue. These could affect the survivin heterodimerization along with its various splice variants and subsequently may result in different roles in apoptosis process and could explain the diversity of survivin functions [21].

The study on serum survivin conducted by Shu-Xia et al. [22] showed no significant difference in the serum survivin levels between OSCC and non-cancer patients, though there were significantly higher levels of survivin detected in the saliva and tumor exfoliated cells of OSCC as compared to the non-cancer patients. One possible explanation for this result could be due to the marker being sequestered at the site of malignant and premalignant lesion and can account for survivin not being detected in the serum of the oral cancer patients as compared to its high levels in the tumor and peritumor fluids as seen in the above study. Since the human survivin ELISA kit used in the present study was specific for the major/wild survivin isoform (survivin wt) and did not detect the other isoforms/splice variants, we feel that the study enabling identification of individual splice variants of survivin will have a much more prognostic value as compared to the present study that evaluated the wild form alone. Adding to the above possibilities is the functional complexity of survivin which has not yet been completely understood in either the normal or tumor cells, despite of the research that has been going on since the past two decades making it difficult to give closing remarks on the role of molecule in cancer or targeted therapy [23].

The studies carried out on serum survivin in other malignancies have shown diversifying results that could be accounted by the characteristic tumor biology of different malignancies. While in some malignancies like pancreatic ductal adenocarcinoma the serum survivin level was highly elevated as compared to the healthy controls and has a negative impact on the prognosis of the patients [24], in malignancies such as melanoma not only there was no significant difference found between the serum survivin levels of the controls and malignancy patients, but also the serum survivin level had increased significantly following the dacarbazine based cytotoxic cheamotherapy [25].

Our study suggests that the measurement of free, wild form of circulating survivin do not correlate with the advancing clinical stage and probably has no role in predicting the prognosis of oral cancer or the malignant transformation potential of oral submucosal fibrosis per se. It would be useful to detect all the isoforms of circulating survivin in the serum, saliva and tissues for a better understanding of its significance in pathogenesis and establishment of oral cancer.

Compliance to Ethical Standards

Conflict of interest

No potential conflicts of interest were disclosed.

Ethical Standards

The study was approved by the ‘Ethics Committee’ of All India Institute of Medical Sciences and Informed consent was obtained from all participants.