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Abstract

BACKGROUND:

The Indian subcontinent has the highest incidence and prevalence of oral squamous cell carcinoma (OSCC). Inflammation and apoptosis are two emerging hall marks of cancer that might play a significant role in tumorigenesis and metastasis. Concurrent expression of proinflammatory cytokine (IL-1 $\beta$ ) and executioner caspase (Caspase-3) in same OSCC tissue samples has not been reported in an Indian population.

OBJECTIVES:

The present study aimed to evaluate the immunohistochemical expression of IL-1 $\beta$ and Caspase-3 in same OSCC tissue samples with clinicopathological correlation and survival analysis in Indian population.

METHODS:

A retrospective study was conducted utilizing 40 formalin fixed paraffin embedded histologically diagnosed cases of OSCC comprising of 20 metastatic OSCC and 20 non-metastatic OSCC.

RESULTS:

Increased expression of IL-1 $\beta$ and Caspase-3 were observed in metastatic OSCC. Correlation of expression of IL-1 $\beta$ and Caspase-3 with clinicopathological parameters revealed a significant association between these markers and staging, nodal status and site of the lesion.

CONCLUSION:

Over expression of IL-1 $\beta$ and Caspase-3 was associated with advanced stage and poor survival of the patient. IL-1 $\beta$ overexpression showed significantly lower disease-free survival and disease specific survival as well. Overexpression of IL-1 $\beta$ and Caspase-3 in incisional OSCC biopsies could be considered for predicting metastasis and survival outcome.

Keywords

Caspase-3 Interleukin-1β mouth neoplasms biologic behaviour cumulative survival survival analysis

1. Introduction

Figure 1.

Pie-chart showing the demographic details of OSCC patients considered for the study. A. Age distribution. B. Gender distribution. C. Tobacco status distribution. D. Staging distribution. E. Site distribution. F. Nodal status distribution.

Despite numerous advancements over the years , annually oral cancer accounts for an estimated 275,000 cases globally. India accounts for about 30% of all new cases annually. OSCC has a survival rate $\leqslant$ 50% after 5 years, however prognosis of locoregionally and metastatically advanced OSCC is poor [1, 2]. Early disease detection and identification of prognostic markers that can predict metastasis and survival are imperative as it can result in better treatment outcome [3, 4, 5, 6].

The link between apoptotic proteins such as caspases and cytokines remain a sparsely researched area in oral oncology. The role of caspases in regulating inflammation resulting in malignancy still remains unclear [7, 8, 9]. Despite the fact that caspases act mainly within the cell, the production and activity of caspases is triggered and modulated by external signals via cytokines thus, making these two entities “Inter-connected” in biological processes. It is proposed that cytokines are produced in response to inflammation and serve as an important link between inflammation and cancer through intrinsic and extrinsic effects [10, 11, 12, 13, 14]. Pro-inflammatory cytokine called Interleukin 1 family (IL-1) has been associated with poor prognosis and survival of patients with gastric and colorectal adenocarcinoma [15]. The tumorigenic potential of pro-inflammatory cytokine IL-1 $\beta$ in OSCC is not yet established.

The correlation and interplay between apoptosis and inflammation is not yet investigated in OSCC. Hence the present study aimed to evaluate the expression of IL-1 $\beta$ and Caspase-3 in metastatic and non-metastatic OSCC. This study will be the first of its kind to evaluate both markers on the same OSCC tissue with clinicopathological correlation and survival analysis in an Indian population. Identification of overexpressed IL-1 $\beta$ and Caspase-3 proteins could result in their utilization in targeted therapy of OSCC.

2. Materials and methods

Ethical clearance for conducting the study was obtained from ethics committee of M.S. Ramaiah University of Applied Sciences (Ref: UECHT/2016-18/PGDT/03).

2.1 Selection of cases

Data were collected using a pre-formatted data collection form. For each case, the corresponding information on age, gender, tobacco status, tumour size, nodal status, metastases (TNM) and follow-up data of five years were collected from clinical records Fig. 1. The clinico-pathological features were classified according to American Joint Committee on Cancer (AJCC) designated staging by TNM classification system. The following criteria was applied for selection of cases. Histopathologically diagnosed incisional biopsies of primary OSCC (metastatic and non-metastatic) were considered having complete clinical data record.

Recurrent cases of OSCC were excluded. Slovin’s formula was applied to the current study to determine sample size, which is expressed as $n=N/(1+Ne2)$ where $n=$ Number of samples, $N=$ Total population and $e=$ Error tolerance level. Hence, sample size was determined as 55 using formula; $n=$ number of cases selected, $N=$ total number of diagnosed cases (75), e (error) $=$ 0.05 (giving an alpha level of 0.05) at confidence level 95%. Hence, 20 samples each of metastatic and non-metastatic groups and 10 samples of normal oral mucosa were included for the study.

2.2 Tissue specimens

A total of 40 formalin fixed paraffin embedded (FFPE) histologically diagnosed cases (20 metastatic and 20 non-metastatic OSCC) were retrieved from archives of the Oral Pathology and Microbiology Department, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences, Bangalore. Normal healthy oral mucosa (NOM, $n=$ 10) were taken as control tissues while poorly differentiated OSCC (PDOSCC) was considered as positive control for IL-1 $\beta$ and Caspase-3. Normal human kidney and tonsil tissue were taken as control sections for IL-1 $\beta$ and Caspase-3 to standardize the IHC staining procedure (to eliminate false positive reactions) as per the manufacturer’s protocol.

2.3 Tissue processing and immunohistochemistry

2.3.1 Deparaffinizing and rehydrating

FFPE tissue specimens measuring 4 $\mu$ m-thick were obtained using a semi-automatic microtome. Deparaffinization of tissue sections were done twice using 100% xylene for a 10-minute duration. The slides were subjected to the descending grades of ethanol at (100%, 95%, 80% and 75%) for 5 minutes followed by rehydration in phosphate buffered saline (PBS) for 15 minutes.

2.3.2 Antigen retrieval and enzyme blocking

All slides and antigen retrieval buffer, pH 6.0 (Pathnsitu Cat# PS007, Hyderabad, India) were held in coplin jars in a water bath set to 92 ${}^{\circ}$ C and incubated for 25 minutes. Sections were incubated for 5–10 minutes with PolyExcel 3% H ${}_{2}$ O ${}_{2}$ which results in quenching of any endogenous peroxidase enzyme and rinsed in PBS for 5 minutes.

Figure 2.

Photomicrographs of control tissue used for standardization A. Section of kidney stained with IL-1 $\beta$ antibody showing a clear background (indicates absence of non-specific staining), (IHC, x100). B. Section of tonsil stained with Caspase-3 antibody showing a clear background (IHC, x100). C. Normal oral mucous membrane exhibiting negative staining for IL-1 $\beta$ antibody (IHC, x100). D. Normal oral mucous membrane demonstrating negative staining for Caspase-3 antibody (IHC, x100). E. OSCC tissue revealing intense cytoplasmic positivity for IL-1 $\beta$ antibody (IHC, x100). F. OSCC tissue revealing high cytoplasmic positivity for Caspase-3 antibody (IHC, x100).

2.3.3 Primary antibody staining

The sections were incubated with anti-mouse monoclonal primary antibody (IL-1 $\beta$ (11E5): sc-52012, 1:60 dilution and Caspase- (31A1067): sc-56053, 1:60 dilution) Santa Cruz biotechnology, California, USA, for each antibody in humidifying chamber with immune wash buffer (Pathnsitu Cat# PS006, Hyderabad, India) for 1 hour at 92 ${}^{\circ}$ C in multi epitope retrieval system. The dilution of the antibody was standardized prior to staining with tonsil and renal tissue for Caspase-3 and IL-1 $\beta$ respectively. Sections were then rinsed in immune PBS wash buffer for 10 minutes at room temperature. Sections were incubated with the PolyExcel target binder for 10 minutes, rinsed with PBS followed by incubation with mouse monoclonal horse radish peroxidase labelled secondary antibody at 37 ${}^{\circ}$ C for 1 hour then rinsed with PBS thrice for 10 minutes at room temperature. Staining was completed by 5–10 minutes incubation with 3, 3’-diaminobenzidine (DAB) substrate – chromogen which results in a brown-colored precipitate at the antigen site. The sections were counterstained with Harris haematoxylin for 5–10 minutes and washed in running water for 5–7 minutes. Finally, the sections were dehydrated by using increasing concentrations of alcohol (50%, 60%, 70%, 80% and 100%) and cleared in xylene for 5 minutes at room temperature followed by placement of coverslip with mounting medium distrene dibutyl phthalate xylene. A negative control where the primary antibody was omitted was included for both antibodies and showed no staining.

Figure 3.

Photomicrographs of OSCC A. Section of non-metastatic OSCC tissue showing invading tumor islands (H&E stain, x100). B. Non-metastatic OSCC stained with antibody to IL-1 $\beta$ showing light cytoplasmic staining (IHC, x100). C. IL-1 $\beta$ showing light cytoplasmic staining at higher magnification (IHC, x200). D. Section of Metastatic OSCC tissue showing invading tumor islands (H&E stain, x100). E. Metastatic OSCC stained with antibody to IL-1 $\beta$ showing moderate cytoplasmic staining (IHC, x100). F. IL-1 $\beta$ showing moderate cytoplasmic staining at higher magnification (IHC, x200). G. Section of Metastatic OSCC tissue showing invading tumor strands (H&E stain, x100). H. Metastatic OSCC stained with antibody to IL-1 $\beta$ showing intense cytoplasmic staining (IHC, x100). I. IL-1 $\beta$ showing intense cytoplasmic staining at higher magnification (IHC, x200).

2.3.4 Interpretation and scoring

Immunohistochemical results were semi quantitatively evaluated in a blinded pattern by two observers (PS and DA), at a magnification of x100 and x400 using Olympus Optical Microscope BX53F2, Tokyo, Japan. Any discrepancy in the scores was solved by a group consultation with third observer (SVS) utilizing penta-head Olympus Optical Microscope U-MDOB3, Tokyo, Japan. The photomicrographs were captured with a Jenoptik Progres Gryphax Arktur USB 3.0 microscope camera, Jena, Germany. For both Caspase-3 and IL-1 $\beta$ brown cytoplasmic staining of the cells was considered as positive. Average staining intensity for Caspase-3 was scored as follows (Pei-Feng Liu et al. 2017): 0 – no staining, 1 – mild, 2 – moderate and 3 – strong. The percentage of stained cells at each intensity level was graded as 0 (5%), 1 (5–10%), 2 (26–50%), 3 (51–75%) and 4 ( $>$ 75%). The intensity score and percentage of positive cells were then added to produce the final scores with range of 0–7 which was further scored as 0 - negative/absent, 1–2 – mild, 3–4 – moderate and 5–7 – strong. For IL-1 $\beta$ the average staining intensity was scored as follows (Alexander et al., 2003): 0 to 4, 0 – no staining, 1 – faint staining, 2 – light staining, 3 – moderate staining and 4 – intense staining. The scores were recorded and subjected to statistical analysis. The clinicopathologic parameters such as age, gender, smoking habit, tumour size, nodal status, metastases (TNM), tumour grade and follow-up data were also recorded and subjected to statistical analysis using the Statistical Package for the Social Sciences (SPSS) software version 20.0, IBM.

Figure 4.

Photomicrographs of OSCC A. Section of non-metastatic OSCC tissue showing invading tumor islands with dysplasia (H&E stain, x100). B. Non-metastatic OSCC stained with antibody to Caspase-3 showing low cytoplasmic expression (IHC, x100). C. Caspase-3 showing low cytoplasmic expression in non-metastatic OSCC at higher magnification (IHC, x200). D. Section of Metastatic OSCC tissue showing invading tumor islands with keratin pearls (H&E stain, x100). E. Metastatic OSCC stained with antibody to Caspase-3 showing high expression (IHC, x100). F Caspase-3 showing high expression in metastatic OSCC at higher magnification (IHC, x200). G. Section of Metastatic OSCC tissue showing invading tumor strands and cords (H&E stain, x100). H. Metastatic OSCC stained with antibody to Caspase-3 showing high expression (IHC, x100). I. Caspase-3 showing high expression in metastatic OSCC at higher magnification (IHC, x200).

3. Results

Following meticulous standardization and obtaining of negative background staining using kidney and tonsil tissue (Fig. 2A and B), the IHC procedure was initiated. NOM taken as healthy controls were immunonegative for IL-1 $\beta$ and Caspase-3 staining (Fig. 2C and D), while the positive control PDOSCC tissue showed cytoplasmic immunopositivity for antibodies to both IL-1 $\beta$ and Caspase-3 (Fig. 2E and F).

3.1 IL-1 $\beta$ expression in non-metastatic and metastatic OSCC

Brown cytoplasmic immunostaining was observed in tumour cells of OSCC positive control tissue for IL-1 $\beta$ . Among 20 non-metastatic OSCC cases, 17 (81%) cases showed a positive cytoplasmic immunoreactivity for IL-1 $\beta$ and 3 (19%) cases were negative. Among the 20 metastatic cases of OSCC, positive immunoreactivity was observed in 19 (90%) cases for IL-1 $\beta$ , while 1 (10%) case was negative. Among the 17 non-metastatic positive cases, 4 (24%) cases showed moderate staining intensity, 3 (17%) cases showed light staining (Fig. 3A–C), and 10 (58%) cases showed faint staining. Among 19 metastatic OSCC, 2 (10%) cases showed faint staining, 3 (15%) cases showed light staining, 5(26%) cases showed moderate staining (Fig. 3D–F), 8 (42%) cases showed intense staining (Fig. 3G–I). When IL-1 $\beta$ expression was compared between non-metastatic and metastatic OSCC a statistically significant result was obtained using Mann-Whitney U Test (Table 1).

Figure 5.

Photomicrographs of metastatic OSCC A. Section of metastatic OSCC tissue showing tumor islands and muscle invasion (H&E stain, x100). B. Metastatic OSCC stained with antibody to IL-1 $\beta$ showing intense cytoplasmic staining (IHC, x100). C. IL-1 $\beta$ showing intense cytoplasmic staining in metastatic OSCC at higher magnification (IHC, x200). D. Section of metastatic OSCC tissue showing tumor islands and vascular invasion (H&E stain, x100). E. Metastatic OSCC stained with antibody to Caspase-3 showing high expression (IHC, x100). F. Caspase-3 showing high expression in metastatic OSCC at higher magnification (IHC, x200). G. Section of Metastatic OSCC tissue showing invading tumor islands (H&E stain, x100). H. Metastatic OSCC stained with antibody to Caspase-3 showing high expression (IHC, x100). I. Caspase-3 showing high expression in metastatic OSCC at higher magnification (IHC, x200).

Table 1

Comparison of IL-1 $\beta$ expression in non-metastatic and metastatic groups

Groups	N	Mean rank	Sum of ranks	U	$p$ value
Non-metastatic	20	14.55	291.00	81.0	0.001
Metastatic	20	26.45	529.00

Mann-Whitney U Test, $p$ -value $\leqslant$ 0.05 considered as significant.

3.2 Caspase-3 expression in non-metastatic and metastatic OSCC

Brown cytoplasmic immunostaining was observed in tumour cells of OSCC positive control tissue for Caspase-3. Among 20 non-metastatic OSCC cases, all 20 (100%) cases showed a positive cytoplasmic immunoreactivity for Caspase-3, of which 8 (40%) cases showed a high score, 6 (30%) cases showed intermediate score and 6 (30%) cases showed low score (Fig. 4A–C). In case of metastatic OSCC, 18 (90%) cases showed high score (Fig. 4D–I), and 2 (10%) cases showed intermediate score. None of the metastatic OSCC cases showed low or negative immunoreactivity for Caspase-3. When Caspase-3 expression was compared between non-metastatic and metastatic OSCC a statistically significant result was obtained using Mann-Whitney U Test (Table 2).

Table 2
Comparison of Caspase-3 expression in non-metastatic and metastatic groups

Groups	N	Mean rank	Sum of ranks	U	$p$ value
Non-metastatic	20	12.65	253.00	43.0	0.001
Metastatic	20	28.35	567.00

Mann-Whitney U Test, $p$ -value $\leqslant$ 0.05 considered as significant.

3.3 Pearson’s correlation of IL-1

\beta

expression and Caspase-3 in metastatic OSCC

When analysis of coefficient of correlation was executed, expression of IL-1 $\beta$ and Caspase-3 in metastatic OSCC showed a significant ( $p=$ 0.021) correlation (Table 3). High correlation of IL-1 $\beta$ and Caspase-3 expression was observed in metastatic OSCC cases as shown in (Fig. 5A–I). Three metastatic OSCC cases showed an inverse correlation where IL-1 $\beta$ expression was negative while Caspase-3 showed high positivity (Fig. 6A–I).

Table 3
Correlation of IL-1 $\beta$ and Caspase-3 expression in non-metastatic and metastatic Oral Squamous cell carcinoma

Groups	Pearson’s correlation (r)	$p$ value, significance
Metastatic	0.355	0.021, S
Non-metastatic	$-$ 0.062	0.794, NS

$p$ -value $\leqslant$ 0.05 considered as significant.

Table 4

Correlation of IL-1 $\beta$ expression with clinicopathological parameters of OSCC

	$n$ (%)
	No staining $n=$ 5		Faint $n=$ 13		Light $n=$ 6		Moderate $n=$ 8		Intense $n=$ 8		Total $n=$ 40		$p$ value	Pearson’s $\chi^{2}$
Age groups
31–40	0		4	(80)	0		1	(20)	0		5	(100)	0.939	0.004
41–50	2	(15.4)	2	(15.4)	2	(15.4)	2	(15.4)	5	(38.5)	13	(100)
51–60	3	(21.4)	3	(21.4)	3	(21.4)	2	(14.3)	3	(21.4)	14	(100)
61–70	0	(0)	4	(50)	1	(12.5)	3	(37.5)	0	(0)	8	(100)
Gender
Male	2	(11.8)	3	(17.6)	3	(17.6)	4	(23.5)	5	(29.40)	17	(100)	0.445	2.364
Female	3	(13)	10	(43.5)	3	(13)	4	(17.4)	3	(13)	23	(100
Smoking status
Yes	4	(13.3)	7	(23.3)	4	(13.3)	8	(26.7)	7	(23.3)	30	(100)	0.151	6.725
No	1	(10)	6	(60)	2	(20)	0	(0)	1	(10)	10	(100)
Site
Buccal mucosa	4	(20)	11	(55)	3	(15)	2	(10)	0	(0)	20	(100)	0.001	12.018
Tongue	1	(8.3)	1	(8.3)	1	(8.3)	5	(41.7)	4	(33.3)	12	(100)
Other sites	0		1	(12.5)	2	(25)	1	(12.5)	4	(50)	8	(100)
Nodal involvement
$N_{0}$	3	(15)	11	(55)	3	(15)	3	(15)	0		20	(100)	0.001	13.498
$N_{1}$	2	(16.7)	2	(16.7)	2	(16.7)	2	(16.7)	4	(33.3)	12	(100)
$N_{3}$	0		0		1	(12.5)	3	(37.5)	4	(50)	8	(100)
Staging
1	2	(33.3)	1	(16.7)	1	(16.7)	1	(16.7)	1	(16.7)	6	(100)	0.160	1.973
2	2	(12.5)	6	(37.5)	2	(12.5)	4	(25)	2	(12.5)	16	(100)
3	1	(9.1)	4	(36.4)	2	(18.2)	2	(18.2)	2	(18.2)	11	(100)
4	0	(0)	2	(28.6)	1	(14.3)	1	(14.3)	3	(42.9)	7	(100)

Chi square test, $p$ -value $\leqslant$ 0.05 considered as significant.

Figure 6.

Photomicrographs of metastatic OSCC with inverse expression of markers A. Section of metastatic OSCC tissue showing dysplastic features (H&E stain, x100). B. Metastatic OSCC showing negative immuno reaction for antibody to IL-1 $\beta$ (IHC, x100). C. Metastatic OSCC stained with antibody to Caspase-3 showing high expression IHC, x200). D. Section of metastatic OSCC tissue showing tumor islands and vascular invasion (H&E stain, x100). E. Metastatic OSCC showing negative immuno reaction for antibody to IL-1 $\beta$ (IHC, x100). F. Metastatic OSCC stained with antibody to Caspase-3 showing high expression (IHC, x200). G. Section of Metastatic OSCC tissue showing invading tumor islands (H&E stain, x100). H. Metastatic OSCC showing negative immuno reaction for antibody to IL-1 $\beta$ (IHC, x100). I. Metastatic OSCC stained with antibody to Caspase-3 showing high expression (IHC, x200).

3.4 IL-1

\beta

expression and clinicopathological correlation

When IL-1 $\beta$ expression was correlated with clinicopathological parameters (age, gender, site, tobacco status, nodal status and staging), the expression level of IL-1 $\beta$ was significantly associated with site (tongue, $p=$ 0.001). Similarly, a significant association of IL-1 $\beta$ over expression was associated with nodal status ( $p=$ 0.001) (Table 4). Despite, statistical insignificant value ( $p=$ 0.151), the high expression level of IL-1 $\beta$ was associated with patient having history of tobacco status, this could be attributed to the inflammation caused in the tissues due to the penetrative effect of tobacco and its constituents. Likewise, positive correlation existed between expression of IL-1 $\beta$ and staging with statistical insignificant value using Chi square test ( $p=$ 0.160).

3.5 Caspase-3 expression and clinicopathological correlation

When Caspase-3 expression was correlated with clinicopathological parameters statistically significant result was obtained in case of nodal involvement ( $p=$ 0.040) (Table 5). However, high expression of Caspase-3 was associated with staging (stage-III & stage-IV) but was statistically insignificant using Chi square test ( $p=$ 0.181).

Table 5
Correlation of Caspase-3 expression with clinicopathologic parameters of OSCC

	$n$ (%)
	Mild and moderate $n=$ 13		Strong $n=$ 27		Total $n=$ 40		$p$ value	Pearson’s $\chi^{2}$
Age groups
31–40	1	(20)	4	(80)	5	(100)	0.351	1.882
41–50	2	(15.4)	11	(84.6)	13	(100)
51–60	6	(42.8)	8	(57.1)	14	(100)
61–70	4	(50)	4	(50)	8	(100)
Gender
Male	5	(29.4)	12	(70.6)	17	(100)	0.692	0.736
Female	8	(34.7)	15	(65.3)	23	(100)
Smoking status
Yes	9	(30)	21	(70)	30	(100)	0.827	0.381
No	4	(40)	6	(60)	10	(100)
Site
Buccal mucosa	8	(40)	12	(60)	20	(100)	0.267	1.231
Tongue	3	(25)	9	(75)	12	(100)
Other sites	2	(25)	6	(75)	8	(100)
Nodal involvement
$N_{0}$	11	(30)	9	(45)	20	(100)	0.040	4.238
$N_{1}$	0		12	(100)	12	(100)
$N_{3}$	2	(25)	6	(75)	8	(100)
Staging
1	2	(33.3)	4	(66.7)	6	(100)	0.181	1.791
2	6	(18.8)	10	(68.5)	16	(100)
3	4	(9.1)	7	(63.9)	11	(100)
4	1	(14.3)	6	(85.7)	7	(100)

Chi square test, $p$ -value $\leqslant$ 0.05 considered as significant.

3.6 Univariate and multivariate survival analysis

Comparison of clinicopathological parameters (age, gender, site, tobacco status and staging) with cumulative survival was done by univariate analysis using Log-Rank (Mantel-Cox) test (Table 6) and Kaplan-Meier plots (Fig. 7). Correlation of site with the survival rate in OSCC patients showed a statistically significant result for tongue carcinoma ( $p=$ 0.001). There was a reduction in the survival rate of OSCC patients with combined habits (smoking and smokeless) by 50% in 200 weeks. When the stage of OSCC and survival was correlated a statistically significant difference was observed between the stages and survival, advanced stages III and IV correlated with decreased survival rate with a $p$ value of 0.002. Kaplan Meier analysis comparing IL-1 $\beta$ expression with survival rate in OSCC subjects showed decreased survival by 70% in 150 weeks with IHC score 4, whereas patients with IHC score of 1 did not show decrease in survival rate in the complete follow-up period of 300 weeks. A statistically significant difference between IHC (IL-1 $\beta$ expression) scores and survival rate was obtained with $p$ value of 0.012. Kaplan Meier analysis comparing Caspase-3 expression with survival rate in OSCC subjects showed decreased survival by 30% in 170 weeks with IHC score of 3. A statistically significant difference between IHC (Caspase-3 expression) scores and survival rate was obtained with $p$ value of 0.002. The DFS and DSS of the patients were calculated using Multiple Cox’s regression analysis (Table 7). Patients with positive expression levels of IL-1 $\beta$ had significantly shorter DSS ( $p=$ 0.045) and DFS ( $p=$ 0.045) compared to patients with low and negative expression, whereas, Caspase-3 immunopositivity was not significantly associated with DSS ( $p=$ 0.396) and DFS ( $p=$ 0.394).

Table 6
Comparison of clinical variables/Clinicopathological Parameters with the cumulative survival using univariate analysis [Log-Rank (Mantel-Cox) test]

Clinicopathological

parameters

\chi^{2}

Degrees of freedom

p

value

Age

2.202

0.532

Gender

0.720

0.396

Site

32.043

0.001

Tobacco status

2.514

0.113

Stage

14.574

0.002

IL-1

\beta

12.769

0.012

Caspase-3

3.633

0.002

Kaplan-Meier method, $p$ -value $\leqslant$ 0.05 considered as significant.

Figure 7.

Kaplan-Meier plots comparing the clinicopathological parameters with cumulative survival. (A) Age group, (B) Gender, (C) Site, (D) Tobacco status, (E) Staging, (F) IL-1 $\beta$ expression and (G) Caspase-3 expression.

Table 7

Correlation of expression levels of IL-1 $\beta$ and Caspase-3 for disease-specific survival and disease-free survival of OSCC patients by Multiple Cox’s regression analysis

Variables	Disease-specific survival	Disease-free survival
	Crude hazard ratio (95% CI)	$p$ value	Crude hazard ratio (95% CI)	$p$ value
IL-1 $\beta$	1.673 (1.033–2.708)	0.036	1.629 (1.011–2.624)	0.045
Caspase-3	1.689 (0.503–5.668)	0.396	1.691 (0.506–5.655)	0.394

Multiple Cox’s regression, $p<$ 0.05-significant.

4. Discussion

One of the major causes for increased incidence of OSCC in India is habit related (smoked or smokeless tobacco) which leads to 8.4 times higher risk than that of patients without habit [16, 17, 18, 19, 20]. For patients without lymph node metastasis, the corresponding survival probabilities are 95%, 86% and 86% for first, second and fifth year respectively, for patients with metastasis it drastically drops to 71%, 52% and 44% [21, 22].

It is advocated that NF- $\kappa$ B regulates tumour angiogenesis through VEGF, a known angiogenic factor. IL- 1 $\beta$ is considered as one of the important stimuli for expression of such angiogenic factors [23, 24, 25, 26, 27, 28]. The present study hypothesizes to evaluate the interplay between the proinflammatory cytokine (IL-1 $\beta$ ) and executioner Caspase-3 in OSCC with its clinicopathological implication and survival analysis with DFS and DSS. Few studies in literature correlate expression of Caspase-3 with clinicopathological parameters and survival in OSCC with none utilizing tissues from the Indian population. There has been no studies that have addressed correlation between expression of IL-1 $\beta$ in metastatic and non-metastatic OSCC tissue samples. Literature review lacks evidence correlating the expression of IL-1 $\beta$ and survival in OSCC. According to our knowledge and literature review the present study is the first of its kind to investigate IL-1 $\beta$ and Caspase-3 expression concurrently in the same OSCC tissue with clinicopathological and survival analysis.

When expression of IL-1 $\beta$ and Caspase-3 was evaluated in metastatic and non-metastatic OSCC a significant correlation was observed among these groups. Expression of both markers were high in metastatic OSCC than that of non-metastatic cases with a significant $p$ value of 0.001. The findings of the current study were in accordance with Liu et al. who reported that over expression of IL-1 $\beta$ supported the metastatic progression of prostate cancer cell line [29]. In a study conducted by Sun et al., 62.5% of hepatocellular carcinoma with metastasis were highly expressive for Caspase-3 [30]. Pearson’s correlation was employed to analyse co-relation of expression of IL-1 $\beta$ and Caspase-3 in metastatic OSCC. A significant co-relation between the markers were observed with $p$ value 0.021. The results obtained support the role of IL-1 $\beta$ and Caspase-3 in the metastatic potential of OSCC. The results of the current study also support the statement that IL-1 $\beta$ and Caspase-3 can be considered as independent markers to predict metastasis in OSCC. The tumour microenvironment has a profound effect on tumorigenesis promoting angiogenesis and metastasis. Dysregulation of IL-1 $\beta$ leads to tumour invasion and metastasis. It induces several chemokines which interact with tumour cells to produce angiogenic and lymphangiogenic factors favouring nodal metastasis.

On analysing the clinicopathological parameters with IL-1 $\beta$ expression, site ( $p=$ 0.001) and nodal metastasis ( $p=$ 0.001) were significantly associated with overexpression of IL-1 $\beta$ . The tongue is constantly in contact with carcinogens released from tobacco and other substances present in saliva, added to this the lingual surfaces of the posterior teeth contribute to trauma and inflammation. This might be the reason for correlation of high expression of IL-1 $\beta$ with site and nodal involvement. So far there has not been any study reported in literature comparing clinicopathological parameters with IL-1 $\beta$ expression in OSCC as the present study is a first of its kind. While correlating clinicopathological parameters with Caspase-3 expression significant association existed for nodal involvement, indicating high expression of Caspase-3 with lymph node metastasis in OSCC ( $p=$ 0.040). Increased expression of Caspase-3 in metastatic OSCC also signifies the relation of Caspase-3 with external stimuli in the form of carcinogens, tissue damage or tissue hypoxia, where the caspases can induce or activate mitogenic signals in dying cells which in turn begin to proliferate. This phenomenon or function of caspase is utilized by cancer cells that enable their survival, proliferation and metastasis [31].

The clinicopathological parameters influence treatment outcome and survival. An insight about the distinct clinicopathological entities enables appropriate management of the patient with better treatment outcome [32, 33, 34, 35, 36, 37]. In the present study, comparison of clinical variables with the cumulative survival using univariate analysis revealed significant association between site ( $p=$ 0.001) and staging of tumor ( $p=$ 0.002). Poor prognosis and decreased survival rate were noted significantly in OSCC of tongue followed by buccal mucosa and gingivobuccal mucosa. One has to bear in mind that smokeless tobacco lesions are common in buccal mucosa /sulcus region where as smoking tobacco lesions are in lip and commissures. Poor prognosis of tongue carcinoma might be due to accumulation of saliva containing carcinogens, poor oral hygiene and chronic irritation (denture related or sharp cusp) that may enhance the risk of these sites [38, 39]. Due to increased vasculature and lymphatic drainage risk of metastasis in cervical lymph node is increased in tongue cancer. Male patients with an age range between 61–70 years showed decreased survival rate by 40% in comparison with females below 60 years. The possible reason for declined survival rate in this age group might be due to accumulation of mutations, decreased DNA repair mechanism resulting in a reduced immune surveillance against cancer cells. However, the results were not statistically significant between age group and survival. A lower survival was associated with combined tobacco habit (smoked and smokeless). A combined habit will increase the permeability of the oral mucous membrane to assault by carcinogens. It could also contribute to accumulation of mutations and accelerated tumorigenesis. However, the values were not statistically significant between combined habit and survival. A significant association was also found between the stage of OSCC and survival ( $p=$ 0.002). An association of nodal metastasis contributes to a higher overall staging level. As the tumour increases in size the advancing tumour cells may infiltrate the adjacent structures and might be associated with lymphatic spread leading to decreased survival rate in patients with stages III and IV. Similar clinicopathologic correlation data was reported by Peixoto et al. on analysis of survival rates on prognostic factors among patients with OSCC [42] and Tirelli et al. who reported the prognosis of oral cancer and its comparison with AJCC staging [43]. The present study has considered site as a clinicopathologic parameter as each site in the oral cavity has its unique anatomic relation with surrounding structures.

When expression of IL-1 $\beta$ was correlated with survival in OSCC patients a significant relation was found ( $p=$ 0.012). Increased expression of IL-1 $\beta$ revealed diminished survival rate with decreased overall 5-year survival leading to poor prognosis. Direct interaction between IL-1 $\beta$ and tumour cells, and macrophages produced due to inflammation increases the production of angiogenic factors resulting in metastasis. This hypothesis explains the decreased survival of patient with high expression of IL-1 $\beta$ . The present study has revealed for the first time, the direct relation of IL-1 $\beta$ overexpression and poor patient survival in OSCC. The above finding indicates that IL-1 $\beta$ could be used as a potential factor to predict tumour progression and poor prognosis in OSCC. Expression of Caspase-3 showed a significant correlation with survival in OSCC ( $p=$ 0.002). Increased expression of Caspase-3 showed decreased overall 5 years survival of OSCC patients. Similar findings were obtained by Hu et al. who stated that an elevated Caspase-3 expression is associated with shortened overall survival in several cancer types (gastric cancer, cervical cancer and ovarian cancer) [44]. Nakopoulou et al. investigated the immunohistochemical expression of Caspase-3 and found it to be an adverse indicator of clinical outcome in human breast cancer [45]. Only 2 studies have thus far investigated the correlation of Caspase-3 overexpression and survival scores in OSCC (Liu et al. and Huang et al.) [46, 47]. The present study has reported these findings for the first time in tobacco habit associated OSCC tissues in the Indian population.

In cancer terminology, DFS is the time period from the primary treatment of cancer to the time the patient survives without any sign and symptoms of cancer. DSS is the time period from the diagnosis of cancer until the time of patient’s death in a defined period of time. Patients who died due to other causes or disease are not included [48, 49, 50]. The current study explored the DFS and DSS based on expression of IL- 1 $\beta$ and Caspase-3. Increased expression of IL-1 $\beta$ correlated with decreased DFS ( $p=$ 0.045) and DSS ( $p=$ 0.036) with statistical significance. The correlation of DFS and DSS with IL-1 $\beta$ expression has not been reported in literature earlier. Increased expression of Caspase-3 was associated with shorter DFS and DSS, however the values were not statistically significant. The association of shorter DFS with high Caspase-3 expression was in accordance with the study by Liu et al. in OSCC tissue samples [46].

5. Conclusion

The present study provides the first evidence in literature correlating expression of IL-1 $\beta$ and Caspase-3 in OSCC with clinicopathological parameters of Indian patients and survival analysis including DFS and DSS. Significant correlation coefficient exists between IL-1 $\beta$ and Caspase-3 in metastatic OSCC, implicating these markers as independent prognostic aids for cervical metastasis and survival. This could help navigate the surgeons for prophylactic neck dissections in OSCC cases overexpressing IL-1 $\beta$ and Caspase-3. Identification of overexpressed proteins in OSCC and blockade of their respective pathways can be utilized in the targeted therapy in the future.

Footnotes

Acknowledgments

The authors thank Dr. Vijayalakshmi Kotrashetti, Professor and Head, Department of Oral Pathology and Microbiology and Dr. Kishore Bhat, Professor, Department of Microbiology, Maratha Mandal’s NGH Institute of Dental Sciences and Research Centre for assisting with the laboratory procedures. The authors thank Dr. Suvi Kanchan for performing statistical analysis.

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Interleukin-1beta and Caspase-3 expression serve as independent prognostic markers for metastasis and survival in oral squamous cell carcinoma

Abstract

BACKGROUND:

OBJECTIVES:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2.1 Selection of cases

2.2 Tissue specimens

2.3 Tissue processing and immunohistochemistry

2.3.1 Deparaffinizing and rehydrating

2.3.2 Antigen retrieval and enzyme blocking

3.1 IL-1 β expression in non-metastatic and metastatic OSCC

Table 2 Comparison of Caspase-3 expression in non-metastatic and metastatic groups

Table 3 Correlation of IL-1 β and Caspase-3 expression in non-metastatic and metastatic Oral Squamous cell carcinoma

3.5 Caspase-3 expression and clinicopathological correlation

Table 5 Correlation of Caspase-3 expression with clinicopathologic parameters of OSCC

Table 6 Comparison of clinical variables/Clinicopathological Parameters with the cumulative survival using univariate analysis [Log-Rank (Mantel-Cox) test]

5. Conclusion

Footnotes

Acknowledgments

References

3.1 IL-1 $\beta$ expression in non-metastatic and metastatic OSCC

Table 2
Comparison of Caspase-3 expression in non-metastatic and metastatic groups

Table 3
Correlation of IL-1 $\beta$ and Caspase-3 expression in non-metastatic and metastatic Oral Squamous cell carcinoma

Table 5
Correlation of Caspase-3 expression with clinicopathologic parameters of OSCC

Table 6
Comparison of clinical variables/Clinicopathological Parameters with the cumulative survival using univariate analysis [Log-Rank (Mantel-Cox) test]