Expression of Neurotensin and Neurotensin Receptor1 in Human Oral Squamous Cell Carcinoma: A Potential Role in Tumor Depth and Nodal Metastasis

doi:10.21203/rs.3.rs-2466427/v1

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Expression of Neurotensin and Neurotensin Receptor1 in Human Oral Squamous Cell Carcinoma: A Potential Role in Tumor Depth and Nodal Metastasis

https://doi.org/10.21203/rs.3.rs-2466427/v1

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World’s sixth most prevalent cancer is Oral squamous cell carcinoma (OSCC) which is a malignant neoplasm developing in the epithelial tissues of the oral cavity. In Pakistan, it is amongst the most commonly reported malignancies in both genders owing to widespread use of risk factors, including betel nut, smoking, gutka etc.

Advance disease, presence of cervical lymphadenopathy and late presentation leads to poor 5-year survival. Certain variables have been studied as predictors of metastasis of which depth of invasion of tumor is directly correlated to it.

Neurotensin, a 13 amino-acid neuropeptide mediates its effects via three receptors, out of which 2 are G-protein coupled receptors NTR1 & 2 alongwith the sole transmembrane domain receptor, NTR3. Aberrant expression and localization has been reported in various tumors and associated with increase tumor depth and aggressiveness.

Methodology:

Histological grading was performed using Broder’s criteria. H&E staining for evaluation of tumor depth analysis. Immunohistochemical staining was used to study expression and localization of NT, NTR1 in each sample.

Results:

Expressional analysis of NTS and its receptor NTR1 in oral squamous cell carcinoma revealed that ~ 62% of all tumor tissues were positive for neurotensin expression. High-affinity receptor for neurotensin NTR1 was expressed in 79% of OC sample tissues. We investigated tumor depth for any correlation with NTS/NTR1 expression. Overall a moderate intensity of expression was observed in all the tissues but it could be observed that as the tumor depth increased from < 5mm to > 10mm, neurotensin expressional profile also increased in terms of IRS scores.

Tumor depth was measured from basement membrane to the deepest point of invasion. Mean tumor depth was estimated to be 15.02mm with majority of the tumors displaying tumor depth greater than 10mm.

Conclusion:

Oral squamous cell carcinoma

nodal metastasis

tumor depth

immunohistochemistry

Neurotensin

Neurotensin Receptors

World’s sixth most prevalent cancer is Oral squamous cell carcinoma (OSCC) which is a malignant neoplasm developing in the epithelial tissues of the oral cavity, comprising the tongue, gingiva, skin, palate, floor of the mouth, retromolar area and buccal mucosa with an estimated 600,000 new cases diagnosed each year, [1, 2]. In Pakistan, the first most prevalent malignancy in males and the second most frequent malignancy in females is oral cancer [3]. This accounts for 15% of all reported cases of cancer, equivalent to 3% identified globally [4]. Oral carcinoma in the cavity accounts for 2% of all malignancies in women and 4% in men [5].

In Western countries, OSCC has a multifactorial etiology with the most prominent triggers being cigarettes and alcohol, and betel quid and areca nut chewing in parts of Asia, which works independently or synergistically [4]. It has been shown that the human papilloma virus (HPV) plays a function in carcinogenesis. However, hereditary heritage often affects the vulnerability of the person to head and neck cancer [6].The average five year survival rate has stayed poor (50–60%) amid tremendous scientific and medical advances attributed to regionally advanced diagnosis and illness, the presence of lymphadenopathic cervical disorder [7].

Neoplasms arise due to compound genetic and epigenetic aberrations that consequentially transmute cells of a particular organ initiating an advanced invasive disease. It is hypothesized that these genetic and epigenetic aberrations are not localized to a specific group of cells, rather they involve all the daughter cells residing in a particular field or surrounding tissues of the tumor. Although not currently transformed completely in invasive disease, these resident cells harbour enough genetic anomalies which may transform them into malignant tumors at any given time in the future.[8]

In order to check whether these factors promote cancer, the unpredictable behavior of oral cancer has contributed to thorough studies. The parameters are divided into epidemiological, histological parameters [9]. Among the factors investigated, the microscopic depth of invasion has been found to interact specifically with lymph node metastasis. Moore et al. described the microscopic measurement of tumour depth as "the deepest tumour invasion from the mucosal surface into the tissue" [10].The rise in tumour depth and metastasis is marked by unregulated proliferation of cancer cells by ECM-degrading enzymes that destroy the metalloproteinase matrix by degrading the extracellular matrix and basement membrane (MMPs). They are the main mediators of cancer metastasis [11]. There are many groups of MMPs in which MMP-9 has been identified as a tumour depth regulator [12].

Neurotensin, a 13-amino-acid neuropeptide typically located in the brain and gastrointestinal tract, plays an essential part in central nervous system processes and controls gastrointestinal functions. Via three receptors, 2 G-protein coupled receptors, NTR1 and NTR2, and a single transmembrane domain receptor, NTR3, NTR3 mediates its effects [1, 13]. In different cancer tissues and cell types, including glioblastoma, colorectal, breast, kidney, thyroid, pancreatic cancer, and aberrant expression has been seen in comparison to the biological activities of NT [11, 15, 17]. The tumourogenic function of NT is primarily performed by NTR1, whose activation promotes cancer proliferation, migration, and invasiveness via multiple signal transduction pathways [14]. The specific antagonist of NTSR1, SR48692, annihilates the in vivo and in vitro NTS results, suggesting possible usefulness as a prospective therapeutic goal [15, 16]. NT was shown to cause MMP-9 expression by activation of PKCC in one gastric cancer sample [11]. To date, the expression and localization of NT and its receptor in OSCC has not been studied. The relation of the oncogenic impact of the NT/NTR1 complex and its important function in tumour invasion depth and nodal metastasis in OSCC will be explored in this report. Immunofluorescence microscopy in both cancerous and surrounding non-cancerous tissues can be used to monitor the expression and localization of NT/NTR1.

Significance Of Study:

Oral cancer, with its rising prevalence and chronically poor survival rates across the past three decades, is a crippling and lethal illness. It is a matter of considerable worry in the globe and a significant danger to Pakistan's public health. The diagnosis of cancer at an advanced stage and increase in depth of tumour increases the chances of cervical lymph nodes metastasis and decreases the chances of survival up to 50%. The discovery of novel violent behavior indicators may set the stage for exploring creative therapeutic objectives for improved survival.

Study Design and Study Setting:

The study design was an analytical cross-sectional. This study was conducted at Histopathology section of Dow Diagnostic Research and Reference Laboratory (DDRRL), Dow University of Health Sciences (DUHS).

Patients were recruited for study from OPD of Oral & Maxillofacial Surgery. Sample taken from OT while the patients were undergoing surgery. 55 tumour samples were obtained from oral squamous cell carcinoma patients (biopsy proven) undergoing wide local excision and neck dissection at Dow University of Health Sciences (DUHS) after obtaining informed consent at the time of surgery. Minimum size of 3(D) x3(L) x2(W) mm for each sample was taken. 10% buffered neutralized formalin was used to fix and store the specimens.

Inclusion Criteria:

Diagnosed patients of Biopsy proven OSCC irrespective of age and gender.

FFPE tissue blocks with at least 70% tumour tissue will be used for immunohistochemistry and tumour depth analysis.

Exclusion Criteria:

Recipients of prior chemotherapy or radiotherapy.
Poorly fixed tissue.
Pregnant and lactating women.

Sample Size Estimation:

The power of the test was calculated to justify the sample size of 45 tissues using G*Power version 3.1.3 software, based on One-way ANOVA with 95% confidence of interval, an effect size of 6.0556 from means with 45 tissues computed from the Neurotensin Expression from tumor depth of ≤ 5 mm, 6-10mm and > 10mm. It was achieved to be more than 99%. The same power of the test was achieved using results from relationship between Receptor Neurotensin Expression and tumor depth with 95% confidence of interval with an effect size of 5.5919 from means with 45 tissues.

Fixation and processing for light microscopy and immunohistochemistry (IHC):

Samples fixed in 10% neutral buffered formalin were processed using automated tissue processor (Medite TPC 15) followed by embedding in formalin in automated paraffin embedding station (TES 99 Medite) to prepare formalin fixed paraffin embedded (FFPE) tissue blocks,

3–4 µm thin slices were sectioned using microtome (SLEE 4062) from FFPE tissue blocks, these sections, following smoothing out in warm water bath maintained at 46–48°C were fixed on separate coated glass slides for hematoxylin and eosin (H&E) staining as well as immunohistochemical (IHC) staining. Slides were labelled with specimen representative number for identification.

Before staining sections were deparaffinized and rehydrated as follows; mounted slides were heated at 60°C in an oven for 30 minutes to dry sections followed by three xylene rinses of 5 minutes each to remove paraffin. Sections were then rehydrated by immersion in reducing concentrations of Ethanol as 100%, 95%, 80%, and 70% respectively, for 2 minutes each, followed by washing under running tap water for one minute to completely remove excess ethanol.

Hematoxylin and Eosin staining (H&E):

For H&E staining, following deparaffinization and rehydration sections were immersed in Harri’s Hematoxylin (Merck, USA) for 5 minutes followed by a minute of washing under running tap water. In the next step, sections were treated with 1% acid water to decolorize whole section except nuclei. Sections were again washed for one minute under running tap water to remove acid water.

Application of 0.1% ammonia water to section was next followed by 3 minutes of washing under tap water, followed by dehydration in graded ethanol from 70–95%. Slides were counterstained with 1% alcoholic Eosin (Merck, USA) for 1–2 minutes, followed again by dehydration in 95% and 100% Ethanol, respectively for 4–5 seconds each to remove excess Eosin. Next step was clearing by 2 minutes immersion in 2 changes of xylene followed by mounting in DPX (distrenedibitylphalatexylene) (BDH, USA).

Tissue analysis and scoring of Immunohistochemistry:

Histological grading was performed on Hematoxylin and Eosin stained slides using Broder’s system and scored I-IV. Grade I = Well- differentiated, Grade II = Moderately differentiated, Grade III = Poorly differentiated, and Grade IV = Anaplastic [18] The expression and localization of NT and NTR1 were evaluated by two independent observers (H.S. and U.B.).

Semi-quantitative analysis was performed using low magnification (x100) to identify areas with even staining. Total of 400 tumor cells were counted at high magnification (x400) to calculate positively stained cells’ percentage [19, 20].

The immunoreactive scores (IRS) were calculated for proteins (NT and NTR1) as a product of positive cells’ percentage with staining intensity. The percentages of positive cells were scored from 0–4: 0 = 0%, 1 = < 10%, 2 = 10–50%, 3 = 51–80%, 4 = > 80%. The staining intensity was measured as 0 = no staining, 1 = weak staining, 2 = intermediate staining, 3 = strong staining. The IRS was grouped into 0 = no expression, 1–4 = low expression, 5–8 = moderate expression, and 9–12 = strong expression [21].

Statistical Analysis:

Tables contain descriptive study of clinicopathological characteristics using window SPSS tools. Microsoft Excel is used to create visual charts with clinicopathological characteristics. Pearson correlation tests are carried out to determine potential associations between neurotensin, the neurotensin receptor and nodal metastasis, size and depth. The p- values are defined by a standard significance of p < 0.5.

Clinicopathological Characteristics Of Cases:

As stated in the methodology section 55 OC biopsy samples were collected. Eight samples were removed from the study owing to inadequate tumor tissue in the obtained biopsy specimens. Hence, the study sample consisted of 47 participants (74.5% male) between the ages of 22 and 84 years (mean age 46.79, SD ± 14.47), other clinical characteristics of study participants and tumors are reported in Table 1.

Table 1

Characteristics of study participants (n = 47)
Characteristics		n (%)
Gender
	Male	35 (74.5)
	Female	12 (25.5)
Tumor site
	Not specified	1 (2.1)
	Buccal mucosa	34 (72.3)
	Tongue	7 (14.9)
	Mandible	1 (2.1)
	Upper lip	2 (4.3)
	Lower lip	1 (2.1)
	Maxilla	1 (2.1)
Tumor size
	T1	6 (12.8)
	T2	13 (27.7)
	T3	15 (31.9)
	T4a	13 (27.7)
Grade
	G1	15 (31.9)
	G2	16 (34.0)
	G3	16 (34.0)
Lymph nodes
	Negative	22 (46.8)
	Positive	53.2)

Expression of NT and NTR1 in OSCC:

Expressional analysis of Neurotensin protein via IHC exhibited an overall mild to moderate reaction with the antibody (staining intensity: 1.81 ± 0.75) expressed by a mean percentage of 62.04% cells. Cumulative values in the form of Immune Reactive Score showed a moderate immune reaction by the cells (IRS = 5.8 ± 3.55). Majority of this reaction was localized to the cytoplasmic region of the cells (87.93%), while a small percentage of protein could be localized to nuclear region (12.77%) (Table 2).

Table 2

Neurotensin measures (n = 47)
Outcome measures	Neurotensin (mean ± SD)
Staining intensity	1.81 ± 0.75
Overall positive cell percentage	62.04 ± 26.07
Cytoplasmic	87.93 ± 21.49
Nuclear	12.77 ± 22.16
IRS	5.80 ± 3.55

Similarly, expressional analysis of Neurotensin Receptor I via IHC exhibited an overall moderate reaction with the antibody (staining intensity: 2.02 ± 0.71) expressed by a mean percentage of 79.06% of cells. Cumulative values in the form of Immune Reactive Score showed a moderate to strong immune reaction by the cells (IRS = 7.11 ± 3.22). Majority of this reaction was localized to the cytoplasmic region of the cells (61.41%), while a modest percentage of receptors could be localized to nuclear region also (38.58%) (Table 3).

Table 3

Neurotensin Receptor 1 Measures (n = 47)
Outcome measures	Neurotensin Receptor (mean ± SD)
Staining intensity	2.02 ± 0.71
Overall positive cell percentage	79.06 ± 16.76
Cytoplasmic	61.41 ± 31.59
Nuclear	38.58 ± 31.59
IRS	7.11 ± 3.22

Tumor Depth Analysis:

Tumor depth was measured from basement membrane to the deepest point of invasion. Mean tumor depth was estimated to be 15.02mm with majority of the tumors displaying tumor depth greater than 10mm (Table 4).

Table 4

Tumor Depth Statistics
Tumor depth (mm)
mean ± SD	15.02 ± 8.73
Tumor depth (n = 47)	n(%)
≤ 5 mm	7 (13.0)
5–10 mm	11 (23.9)
> 10 mm	29 (63.0)

To evaluate significance of changes in tumor depth in relation to other tumor characteristics, it was compared with status of lymph nodes metastasis and a statistically significant positive correlation was observed between the two (p-value = 0.025) (Table 5).

Table 5

Comparison of Tumor depth with lymph node status
		Lymph node Status
		Negative (n%)	Positive (n%)	p-value
Tumor depth (n = 46)	≤ 5 mm	5 (83.3)	1 (16.7)	0.025
	6–10 mm	7 (63.6)	5 (36.4)
	> 10 mm	9 (31.0)	20 (69.0)
p-value calculated using Chi-square/Fisher's Exact test

Correlation of Tumor Depth and Lymph Node Status with NT/NTR1 Expression in OSCC:

Comparison of Neurotensin expression and localization with changes in tumor depth demonstrated a higher mean expression in tumors with ≤ 5mm of depth and tumors with > 10 mm of depth of invasion while tumor that were between 6–10 mm in terms of depth of invasion displayed slightly lower expressional values. In terms of significance Neurotensin localization was observed to have a statistically significant relationship with increasing tumor depth (p-value: 0.002) (Table 6).

Table 6

Neurotensin expression and localization with tumor depth
Tumor depth
		≤ 5 mm	6–10 mm	> 10 mm	p-value
Neurotensin expression
	mean ± SD	60.00 ± 32.78	50.45 ± 27.42	64.82 ± 25.15	0.319
Neurotensin localization
	mean ± SD	90.40 ± 17.09	68.45 ± 35.11	95.00 ± 9.47	0.002
IRS	mean ± SD	5.40 ± 4.45	4.45 ± 2.20	5.93 ± 3.69	0.493
p-value calculated using independent t-test

Evaluation of NT expression and localization with status of neck lymph nodes displayed a trend towards increased mean expressional and localization values in tumor positive lymph nodes, although the no statistical significance could be obtained (NT expression p-value: 0.778, NT localization p- value: 0.066) (Table: 7).

Table 7

Neurotensin expression and localization with lymph node
		Lymph node
		Negative	Positive	p-value
Neurotensin expression
	mean ± SD	59.76 ± 22.71	62.00 ± 29.50	0.778
Neurotensin localization
	mean ± SD	81.09 ± 28.46	93.08 ± 13.12	0.066
IRS	mean ± SD	5.62 ± 3.29	5.44 ± 3.59	0.862
p-value calculated using one-way ANOVA

Although mean expression of NTR1 can be seen increasing with increasing tumor depth from ≤ 5 mm to > 10mm but the correlation is not statistically significant. Localization of NTR1 was observed to increase in cytoplasm with increasing tumor depth demonstrating a statistically significant positive correlation between the two (p-value of 0.023). (Table 8)

Table 8

R.Neurotensin Expression and localization with tumor depth
Tumor depth		R. Neurotensin Expression		R. Neurotensin Localization
	n	mean ± SD	p-value	Cytoplasmic	Nuclear	p-value
	n	mean ± SD	p-value	mean ± SD	mean ± SD	p-value
≤ 5 mm	7	66.00 ± 22.47		42.00 ± 29.49	58.00 ± 29.49
6-10mm	11	75.00 ± 14.14	0.088	43.40 ± 37.55	56.60 ± 37.55	0.023
> 10mm	29	82.65 ± 16.07		70.37 ± 27.34	29.62 ± 27.34

On the other hand, NTR-1 mean localization was found to have increased in the nuclear region of the cells of tumor positive neck lymph nodes, although no statistically significant correlation could be observed (p-value: 0.69) (Table: 9).

Table 9

R.Neurotensin localization with Lymph Node Status
Lymph node		R-Neurotensin Localization
	n	Cytoplasmic	Nuclear	p-value
	n	mean ± SD	mean ± SD	p-value
Positive	26	59.72 ± 30.25	40.28 ± 30.25	0.690
Negative	21	63.60 ± 34.62	36.40 ± 34.62	0.690

Receiver-Operator Characteristic (ROC) curves were plotted for the values of Expression of Neurotensin and Neurotensin Receptor against tumor depth > 10 mm. The Youden Index was computed for best cutoffs of Expression of Neurotensin and Neurotensin Receptor for the tumor depth > 10 mm. The 42.5 (AUC 64.9%, p-value 0.108) was the optimal Expression of Neurotensin and 87.5 (AUC 73.3%, p-value 0.012) was the optimal Neurotensin Receptor cut off for tumor depth > 10 mm where the Index of Youden was highest. (Table 10 and Fig. 1)

Table 10

Expression of Neurotensin and Neurotensin Receptor cut-off for tumor depth (> 10 mm) using receiving operative curve (ROC)
	Cut-off	Sensitivity	Specificity	AUC	p-value
Expression of Neurotensin	42.5	82.5%	46.7%	64.9%	0.108
Neurotensin Receptor	87.5	58.6%	86.7%	73.3%	0.012
AUC: area under the curve

A sequence of essential genetic and molecular modifications that cause deregulation of cell proliferation, adhesion, migration, and invasion are implicated in cancer development, contributing to invasiveness correlated with metastatic spread of malignant tumors. Various molecular pathways and their key elements are being investigated for changes in normal cell functions and sustenance properties which might cause them to swerve towards the path of neoplastic transformation.

The main contributing factors to the higher mortality of patients with OSCC tend to be local tumor recurrence, second primary tumors, and metastases following traditional therapy. Around 60% of patients are affected by local tumor recurrence, and 15–25% develops metastasis.

There are several characteristics of the tumors being studied which may indicate the path taken by the disease in the future or predict disease prognosis. These parameters as postulated by various studies include the size of the tumor at the time of treatment, its depth of invasion, tumor volume, grade of the cellular atypia, involvement of neck nodes, perineural and perivascular invasion, distant metastasis, and status of margins at the time of surgery [22].

Also, there are several genetic changes in terms of mutation and expression that are being investigated to find a particular target for cancer therapy that may result in a tight control or absolute resolution of the disease. We used a similar approach and investigated the expression and localization of a peptide neurotensin that might have a role in oral cancer. Neurotensin is a 13 amino acid peptide found in the hypothalamus and small intestine. There is evidence pointing towards its role in cell proliferation suggesting its role in growth stimulation of several types of cancers via endocrine, autocrine, and paracrine effects [23]. Various studies have demonstrated that NT and its high-affinity receptor NTR1 might have a significant role in the invasion and metastatic potential of the neoplastic tissues and may serve as significant biomarkers for prognostic evaluation of the neoplastic disease in different tissues of the body for instance Shimizu et al identified that neurotensin and neurotensin receptor’s oncogenic role as a prognostic marker and possible therapeutic target in the cases of head and neck squamous cell carcinoma. The results of this article strongly with NTSR1 knock down by small interference RNA.[16, 24]. Similarly, Alifano et al. demonstrated that NTSR1 activation may participate in lung cancer progression and may be used as a new prognostic biomarker for surgically resected stage I lung adenocarcinoma [47]. Other cancers that might have their progression attribute to NTS and its receptors include cancers of the breast, prostate, liver, colon, pancreas, and melanocytes [26, 27, 28, 29, 30–31].

In this research Neurotensin, a tridecapeptide, and its receptor NTR1 was investigated for their genetic changes in human oral squamous cell carcinoma tissues in relation to some clinicopathological characteristics such as tumor size, depth, grade, and neck lymph nodes metastasis.

In terms of demographics, our research is in agreement with other researches performed in this region where the mean reported age were ≥ 50 years with a higher number of patients being male. A higher mean age tends to point towards a poor prognosis as higher age corresponds to a higher number of comorbidities and hence a higher complication rate after the treatment [32, 33]. In comparison to the population of Europe and North America where cigarette smoking and alcohol are the major factors for oral cancer which lead to the development of OC more frequently in the floor of the mouth and ventral surface of the tongue [34], most common site of tumor in our study was buccal mucosa signifying the effects of common habits of the population of this region such as the frequent use of chewable tobacco, betel quid, areca nut, and different combination of these products which are kept in the buccal sulcus for long periods of times.

Expressional analysis of NTS and its receptor NTR1 in oral squamous cell carcinoma revealed that ~ 62% of all tumor tissues were positive for neurotensin expression in contrast to breast cancer where only 30% of the tumors had expressed this molecule signifying its role in pathogenesis of OC. High-affinity receptor for neurotensin NTR1 was expressed in 79% of OC sample tissues in contrast to breast cancer where 91% of the breast cancer tissues expressed this receptor [27]. Higher NT: NTR1 ratio in OC tissues compared to breast cancer tissues indicated higher functioning of this protein in OC lesions. It has been suggested that NTS/NTR1 interactions play a role in tumor progression by causing activation and/or over expression of EGFR and HER2/3 receptors [35] Also, blocking the function of NTS/NTR1 by targeting NTR1 via its antagonist (SR48692) is known to reduce tumor growth in non-small cell lung cancers [36]. Tumor depth is closely associated with regional lymph node metastasis, correspond to higher chances of recurrence and lower survival rates [37]. We investigated tumor depth for any correlation with NTS/NTR1 expression. Overall a moderate intensity of expression was observed in all the tissues but it could be observed that as the tumor depth increased from < 5mm to > 10mm, neurotensin expressional profile also increased in terms of IRS scores (table: 6). Similarly, number of involved nodes by the tumor, extra- capsular spread of tumor, and levels of the nodes involved, are known to be associated with prognosis of the disease [38, 39–40] Comparison of NT expression in tumors that were associated with tumor positive lymph nodes was found to have no statistically significant association between the two indicating that neurotensin may have a role in local disease progression rather than metastatic spread of the OC. To further test this theory we compared the tumor size with NT expression. Tumor size is an important part of the internationally used TNM classification of Oral Cancer, where T1/T2 is referred to as low- risk tumors with good survival rates and T3/T4 referred to as high-risk tumors. Furthermore, increasing tumor size has been associated with cervical lymph node involvement [41, 42], higher recurrences [43, 44], and poor prognosis of the disease [45, 46]. When tumor size was compared to neurotensin expression an overall moderate intensity of expression was observed in the case of all the tumors with no statistically significant correlation. Although, a slight increase in mean NT expression as tumor size increased from T1-T3 indicating that increased tumor size is positively correlated with increasing neurotensin expression (Table 11).

Table 11

Neurotensin expression and localization with tumor size
Tumor size
		T1	T2	T3	T4a	p- value
Neurotensin expression
	mean ± SD	48.00 ± 27.29	63.84 ± 30.28	67.00 ± 24.26	56.15 ± 24.59	0.473
Neurotensin localization
	mean ± SD	79.40 ± 41.63	90.15 ± 15.18	93.00 ± 12.01	82.00 ± 27.37	0.472
IRS	mean ± SD	4.20 ± 2.68	6.00 ± 3.53	6.27 ± 3.95	4.69 ± 2.89	0.493
p-value calculated using one-way ANOVA

Concomitantly, tumor grade according to the degree of differentiation of the tumor tissue was also correlated with the neurotensin peptide expression. It is well established that the grade of the tumor is significantly related to the 5-year survival rate. Kademani et al reported a mean 5-year survival with a 95% confidence interval for grade I to be 54–80%; for grade II, 41–62%; and for grade III 29–70% [46]. In this study grading of tumor differentiation, expression of neurotensin was found to be increasing from grade I to grade III type of disease although there was a slight decrease in the case of grade II as compared to the other two grades (Table 12).

Table 12

Neurotensin expression and localization with tumor grade
Tumor grade
		G1	G2	G3	p-value
Neurotensin expression
	mean ± SD	61.07 ± 27.39	54.37 ± 25.28	67.50 ± 26.52	0.380
Neurotensin localization
	mean ± SD	93.50 ± 10.52	87.06 ± 24.87	83.00 ± 26.36	0.437
IRS	mean ± SD	5.07 ± 2.58	4.75 ± 3.29	6.69 ± 4.02	0.238
p-value calculated using one-way ANOVA

Lastly, Considering localization of the neurotensin it was found most concentrated in the cytoplasm (62.04 ± 26.07) with scarce localization (12.77 ± 22.16) in the nuclear region a similar trend was seen in the case of NTR1 majority of which was found located in the cytoplasm (61.41 ± 31.59), while a lesser concentration was seen in the nuclear region of the cells (38.58 ± 31.59). Significance of this distribution and its effects on cell physiology and pathology remains to be studied.

Our study demonstrated a general trend towards a higher expressional gradient of neurotensin correlating with increasing depth of invasion which is known to be an independent predictor of the OC prognosis. Also, this indicates a potential role of this peptide in determining the aggressiveness of the disease. Our research poses the likelihood that, NTS and NTSR1 may serve as a beneficial predictive marker of OC prognosis, also it may serve as a target for therapeutic interventions in OC cases. Further studies are required with a larger sample size and more sensitive identification tools to establish this relationship and to make it a therapeutic target.

The study’s major limitation is the sample size for evaluation is relatively small and lack of RNA extraction. The study was single center (DUHS), which created difficulty in collecting samples and didn’t represent the entire population. In future the samples should be multicenter.

The genetic implications of NT/NTR1in OSCC needs to be studied further utilizing cell culture and/or animal models to identify its target molecules and role as a therapeutic agent to control metastasis of the disease and improve prognosis.

OSCC: Oral Squamous Cell Carcinoma, IHC: Immunohistochemistry, PBS: Phosphate Buffered Saline, BNF: Neutral Buffered Formalin, NT: Neurotensin, NTR: Neurotensin receptor, IRS: Immunoreactive scores

Ethics approval and consent to participate

This study was conducted after approval from Institutional Review Board (IRB) of Dow University of Health Sciences (DUHS). IRB Number: IRB-1089/DUHS/Approval/2018. All techniques employed in the study were in agreement with the 1964 Helsinki Declaration and the standards approved by the institutional and/or national research committee. Informed consent was obtained in writing from all the research participants.

Consent for publication

Not Applicable.

Availability of data and materials

The data collected and used for this research is the property of Dow University of Health Sciences, whereas the data used in this study is available upon request from the corresponding author.

Competing interests

The authors declare that they have no conflict of interest.

Funding

This is a self-funded research project.

Authors’ contribution

SJ: Contributed to conceptualizing the overall study, data collection, sample processing and drafting of the manuscript.

AHS: Supervised as well as helped in drafting and editing of the manuscript.

SA: Co-Supervised the study in addition to drafting and editing the main manuscript.

HS: Contributed and helped in the assessment of patients with Oral Squamous Cell Carcinoma.

SZ: Contributed to analyzing the results of the study

NM: Co-Supervised the study and contributed to conceptualizing the overall study and drafting, editing of main manuscript.

Acknowledgements

We are highly indebted to all the research participants for their constant co-operation during the study. We are also extremely grateful to Dow Research Institute of Biotechnology and Bio-Sciences for providing us access to all the necessary equipment for this research.

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No competing interests reported.

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Expression of Neurotensin and Neurotensin Receptor1 in Human Oral Squamous Cell Carcinoma: A Potential Role in Tumor Depth and Nodal Metastasis

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Abstract

Figures

Background

Significance Of Study:

Methodology

Study Design and Study Setting:

Inclusion Criteria:

Exclusion Criteria:

Sample Size Estimation:

Fixation and processing for light microscopy and immunohistochemistry (IHC):

Hematoxylin and Eosin staining (H&E):

Tissue analysis and scoring of Immunohistochemistry:

Statistical Analysis:

Results

Clinicopathological Characteristics Of Cases:

Expression of NT and NTR1 in OSCC:

Tumor Depth Analysis:

Correlation of Tumor Depth and Lymph Node Status with NT/NTR1 Expression in OSCC:

Discussion

Conclusion

Limitation/weakness Of This Study And Directions For Future Research

Abbreviations

Declarations

References

Additional Declarations

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