The present study was carried out at the Department of Oral and Maxillofacial Pathology and Microbiology. The study protocol was approved by our Institutional Ethical Committee. The tumours studied were preselected based on the diagnosis and from the biopsy material acquired from last two years from both major and minor SG. The tumours included 40 cases of benign and 50 cases of malignant SG tumours [Table/Fig-1]. The 40 cases of benign tumours consisted of 20 pleomorphic adenoma, 05 monomorphic adenoma, 08 Warthin’s tumours and 07 canalicular adenoma. The 50 cases of malignant tumours consisted of 26 mucoepidermoid carcinoma of which 11 were high grade type, 08 were low grade type and 06 were of intermediate type, 14 adenoid cystic carcinoma and 06 Acinic cell carcinoma, 02 CXPA and 02 Polymorphous low grade carcinoma. Immunohistochemistry was performed on tissues fixed in 10% neutral buffered formalin, paraffin embedded tissue. The sections were cut serially to 5 μm for immunohistochemistry to evaluate expression of XIAP and Ki-67 antigens.

For immunohistochemistry, Peroxidase Detection System (Streptavidin-Biotin Detection System HRP-DAB; Product Code: RE7110K, Novo- castra kit) was employed. Endogenous peroxidase activity was blocked by treating hydrated sections with 3% H₂O₂ in methanol for 30 min. The slides were heated in a microwave oven for 10 min in 0.01M sodium citrate buffer (pH 6.0) for antigen retrieval and bench cooled for 20 min and again the same cycle was repeated. To prevent non-specific reactions, sections were incubated with 10% serum for 10 min. Pre-diluted Ki-67 antibody (clone MIB-1; Product code: N1633, Dako, Denmark) and diluted at 1:50 XIAP antibody (Cell Signaling technology, USA, cat # 2042) were incubated at room temperature in a humidifying chamber for 60 min and then at 4°C overnight. CXPA is generally an aggressive high-grade carcinoma arising in association with a primary or recurrent PA. Thus, known histopathological sections of CXPA revealed good expression of XIAP and Ki-67 antigen which was used as a positive control. One section from each positive control was used during each batch of immunohistochemistry procedure. This was followed by incubation with secondary biotinylated antibody and streptavidin-peroxidase reagent at room temperature in a humidifying chamber for 30 min. Freshly prepared substrate/chromogen solution of 3, 3’ Diaminobenzidine (mixing 5 ml of concentrated DAB in 50 ml of substrate buffer) was used to visualize the antigen–antibody reaction. Finally, the sections were counterstained in Mayer’s hematoxylin.

Assessment of immunohistochemical stained sections were performed by 05 trained histopathologist and average was taken into consideration.

Ki-67 – The cells were considered positive for Ki-67 antigen if there was an intra-nuclear DAB staining (brown colour). All the stained nuclei were scored positive regardless of their intensity of staining. Cells that lacked a clear nucleus were excluded. Minimum of 1000 cells were counted in each section. Tissue sections were scanned at 100 magnification for most heavily labelled Ki-67. The cell counts were made on captured image at 400 magnification with conventional light microscope in 10 randomly selected fields. Ki-67 labelling index (Ki-67 LI) was calculated from the ratio of the number of tumour cells stained by Ki-67 to the total number of tumour cells counted per section.

XIAP – The cells were considered positive for XIAP antigen if there was granular cytoplasmic staining (brown colour) as in agreement to Benjamin et al., [14]. Tissue sections were scanned at 100 magnification for most heavily labelled XIAP. Granular cytoplasmic staining was considered positive and intensity was graded at 400 magnification which were made on captured image with conventional light microscope in four to five fields randomly selected. The intensity was graded as 0 = no positive cells, 1 = 1–33% positive cells (mild), 2 = 34–66% positive cells (moderate), and 3 = 67–100% positive cells (strong).

Group means for XIAP and Ki-67 LI were derived for each group. The data was analysed statistically using SPSS 16.0 version software for Windows, Mann-Whitney U-test and Pearson’s rank correlation analysis test. The level of statistical significance is at p < 0.05.

Ki-67 LI in benign SG tumours found to be ranging from 0.00 to 1.70 and in malignant SG tumours was 7.60 (±5.40). Ki-67 antigen was expressed in 40% of benign and 48% of malignant SG tumours. It was expressed in both epithelial cells as well as myoepithelial cells.

Descriptive statistics for Ki-67 expression between Benign and malignant SG tumours was done. Statistically significant difference was seen with p-value (p = 0.000) for KI-67 expression between benign and malignant SG tumours. Thus, significantly higher LI is seen in the malignant SG tumours than benign SG tumours [Table/Fig-1,2].

XIAP antigen was expressed in 10% of benign SG tumours all of with mild expression and 26% malignant SG tumours of which 61.53% were with mild expression, 30.76% were moderate and 7.69% were showing strong expression. XIAP was expressed in both epithelial cells as well as myoepithelial cells. Descriptive statistics for XIAP expression between Benign and malignant salivary gland tumours was done. Statistically significant difference was seen with p-value (p=0.016) for XIAP expression between group 1 and group 2. Thus, significantly higher XIAP expression is seen in malignant SG tumours than benign SG tumours [Table/Fig-1&3].

Statistically significant positive correlation between Ki-67 count and XIAP expression was noted in benign and malignant SG tumours [Table/Fig-4].

SG tumours are rare and the diagnosis is often not straightforward, as different types of SG tumours may share overlapping features [19]. The majority of salivary gland neoplasms are epithelial in origin with 70% being pleomorphic adenomas. Among all SG tumours, only adenoid cystic carcinoma, mucoepidermoid carcinoma and carcinoma ex pleomorphic adenoma have histologic spectrum that allows significant tumour grading [20–22]. Inspite of numerous attempts towards histopathological grading the correlation of grades with diseases progression remains ambiguous. Therefore a need is felt for specific marker indicating of aggressive nature of any particular tumour.

In the present study, evaluation of XIAP expression in 40 benign SG tumours was done of which 10% showed positive expression and in 50 malignant SG tumours, 26% showed positive expression [Table/Fig-5a-c]. The XIAP expression was localised with small number of acinar, ductal, luminal, basal and myoepithelial cells in benign tumours as compared to that of the malignant tumours [Table/Fig-5a-c]. This is found in agreement with the study by Benjamin et al., [14].

[Table/Fig-5]:

A: Photomicrograph showing XIAP expression in Pleomorphic adenoma (40X)

B: Photomicrograph showing XIAP expression in Mucoepidermoid Carcinoma (40X)

C: Photomicrograph showing XIAP expression in CXPA (40X)

The mechanisms of apoptosis are highly conserved in a plethora of species and they include a series of processes. Usually, apoptosis can be divided into three distinct phases: the initiation, the effector, and the execution phase. When outer stimuli initiate cellular apoptosis by different pathways, including death receptor-mediated and stress-dependent triggers, the imbalance between activators and IAP of apoptosis appears. After initiation, the activated caspases family and changes in mitochondrial outer membrane permeability finally result in cellular apoptosis [23]. Suppression of apoptotic process by inhibiting procaspase activation and the catalytic activity of mature caspases is mainly carried out by IAP family. As an important member of IAP family which was originally identified in baculovirus, XIAP suppresses apoptosis via inhibition on caspase-3 and -7 [24]. Thus increase in XIAP expression indicates more aggressive phenotypes.

In present study, in benign SG tumours, only pleomorphic adenoma was qualitatively graded mild [Table/Fig-5a]. In malignant tumours, maximum expression was found with CXPA which was qualitatively graded from moderate to strong; with high grade mucoepidermoid carcinoma qualitatively graded from mild to moderate [Table/Fig-5b]. CXPA was the only malignant tumour showing strong grading [Table/Fig-5c]. This was found in agreement with the study by Benjamin e al., [14]. This indicated the blocking of the downstream portion of the apoptosis pathway from multiple stimuli thereby inhibiting both the initiation and execution phases of the caspase cascade crucial to the demise of malignant cells. The other SG tumours lacking XIAP expression might be due to early stages of malignancy process thus lacking increased amount of aberrant protein and of small sample size of those particular subset. An alternative interpretation, for example, that staining heterogeneity could be, to some degree, fixation-dependent, is not categorically ruled out in the present study.

Thus XIAP expression is more in carcinomas indicating a defective apoptotic pathway in malignant neoplasm. Abnormalities in the expression of XIAP, P53, and Her-2/c-erB-2 may represent components of a complex pathogenesis involving multiple molecular pathways or may be secondary events to a single initial pathway resulting in tumour development [9–11,14,19].

Ki-67 antigen is present in all the active parts of cell cycle except G0 phase and reaches its peak during the G2 and M phases [25]. Ki-67 expression is increased with the cell cycle dysregulation. In previous some studies, Ki-67 expression was detected negative in benign SG tumours [26,27] while others noted sporadic Ki-67 expression ranging from 0.00 to 2.81 in the same [19,28]. Recently, Vargas et al., [19] noted Ki-67 LI of 8.38 (±7.24) in malignant salivary gland tumours.

In the present study, Ki-67 expression was evaluated in 40 benign SG tumours, 40% of them showed positive expression and in 50 malignant SG tumours, 48% of them showed positive expression [Table/Fig-6a-c]. Almost all the ductal and sheet pattern of malignant epithelial cells was positive for Ki-67 expression, which was suggestive of high biological aggressive behaviour of tumour with poor prognosis. Ki-67 labelling index was increased in malignant salivary tumour as compared to benign SG tumours indicating high proliferative activity (e.g. cells committed to cell cycle). This was found in agreement to the study by Vargas et al., and Lazzaro et al., [19,26].

[Table/Fig-6]:

A: Photomicrograph showing Ki 67 expression in Pleomorphic adenoma (40X)

B: Photomicrograph showing Ki67 expression in Mucoepidermoid Carcinoma (40X)

C: Photomicrograph showing Ki67 expression in CXPA (40X)

When individual and overall correlation was drawn between XIAP and Ki-67 expression in benign and malignant salivary gland tumours, it was found to be statistically significant. Thus, this present finding not only goes in the favour as the individual function of the marker used but also when they are correlated with each other. As the expression of XIAP protein, an antiapoptotic marker increases, tumour cell death is inhibited or decreased resulting in uncontrolled cell proliferation than normal. Ki-67 one of the essential protein necessary for cell proliferation indicates that the antiapoptotic activity and proliferative activity of the tumour cells is much less in benign salivary gland lesion and is significantly much higher in malignant salivary gland neoplasm.

In general, as tumours become more undifferentiated they acquire cellular alterations that may provide survival and growth advantages and increase clinical aggressiveness. In particular, the enhanced ability to resist apoptosis could increase the likelihood of a malignant cell surviving in stressful or distant microenvironments; including exposure to radio- or chemotherapy and localization to metastatic sites [6]. This is the first study carried out in such a large cohort of salivary glands tumours and third one in oral neoplasm using XIAP marker [14,29]. Also, XIAP-targeting drugs flavonoid phenoxodiol, which has been shown to reduce XIAP and potentiate the action of chemotherapeutic agents in vitro, restored chemo responsiveness in a subpopulation of patients with recurrent ovarian carcinoma [30]. The present findings raise the possibility that XIAP expression could be one of the factors responsible for the ineffectiveness of these therapies in salivary gland tumours.

The study was designed using two markers that is one a proliferative and other IAP. It was anticipated that there would be direct relationship between their expression that is increase in one would cause increase in other. Both the markers showed increase expression from benign to malignant SG tumours. Both experimental and clinical studies suggest that reversal of XIAP actions may enhance therapeutic efficacy. For example, rituximab-induced decrease of XIAP protein levels has been shown to sensitize chronic lymphocytic leukaemia cells to the cytotoxic effects of chemotherapy in vivo. Reversal of radiation resistance has been demonstrated in tumour cells transfected with an adenoviral XIAP antisense vector. XIAP-targeting drugs might similarly hold promise form treatment of unresectable, widely metastatic, or drug-resistant SG malignancy. Further continuation of this study is needed for other SG tumours with large sample size for evaluating the expression of XIAP protein and its role in future target therapy.