CONFLICT OF INTEREST:

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1 LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

MEDICAL ACADEMY FACULTY OF MEDICINE DEPARTMENT OF BIOCHEMISTRY

IBRAHIM BAHROU

ANALYSIS OF INTERLEUKIN-1 AND INTERLEUKIN-6 LEVELS IN THE SALIVA AND SERUM OF ORAL SQUAMOUS CELL CARCINOMA PATIENTS

Requirements for the Degree Master of Medicine

Lithuanian University of Health Sciences

Supervisor:

Prof. dr. Rasa Baniene

Kaunas 2018

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SUMMARY

Author name:Ibrahim Bahrou

Research title: analysis of Interleukin-1β levels in saliva and serum of oral squamous cell carcinoma patients and analysis of Interleukin 6 levels in saliva and serum of oral squamous cell carcinoma patients.

Aim. to analyze and compare the changes in Interleukin-1 and Interleukin-6 concentrations in Saliva and Blood Serum samples of Oral Squamous Cell Carcinoma and Control group patients

Objectives.To investigate and compare the levels of Interleukin-1β in Saliva and Blood Serum of Oral Squamous Cell Carcinoma patients and Control group patients; to investigate and compare the Levels Interleukin-6 in Saliva and Serum of Oral Squamous Cell Carcinoma patients and Control group patients; To make a vulnerable statisitical analysis of the results.

Methods. Saliva and serum samples were collected from 16 oral floor squamous cell carcinoma (OSCC) patients and from 29 control group patients from the Department of Maxillofacial surgery of Kauno Klinikos.An experimental study was done in the Department of Biochemistry of LUHS. For data analysis student-t test was used.

Results. The mean level of interleukin-1 in the saliva of OSCC patients was (1297.9 pg /ml), which was found to be significantly higher than its level in control group patients (531.6 pg/ml). The mean level of interleukin-1 in the serum of OSCC patients was (4.275 pg/ml), which was found to be significantly higher than its level in control patients(3.45 pg/ml). The mean level of interleukin-6 in saliva of OSCC patients was (353.27 pg/ml) which is significantly higher than its level in control group patients (160.274pg/ml). The mean level of interleukin-6 in the serum of OSCC patients was (7.289 pg/ml) which shows no significant difference than its level in control patients (3.387 pg/ml).

Conclusion. IL-1β level in the saliva and serum of OSCC was significantly higher than in control group. IL-6 level in saliva of OSCC was significantly higher as compared to control group, while there was no significant difference between IL-6 concentration in serum of OSCC and control group.

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SANTRAUKA

Darbo autorius: Ibrahim Bahrou

Darbo pavadinimas: Interleukino-1 ir interleukino-6 koncentracijų pokyčiai sergančių burnos plokščialąsteline carcinoma seilėse ir kraujo serume.

Aim: Įvertinti ir palyginti interleukino-1 ir interleukino-6 koncentracijų pokyčius sergančių burnos plokščialąsteline karcinoma ir kontrolinės grupės seilių bei kraujo serumo pavyzdžiuose.

Uždaviniai: Įvertinti interleukino-1 koncentracijos pokyčius sergančių burnos plokščialąsteline karcinoma ir kontrolinės grupės seilių ir kraujo serumo pavyzdžiuose; įvertinti interleukino-6 koncentracijos pokyčius sergančių burnos plokščialąsteline karcinoma ir kontrolinės grupės seilių ir kraujo serumo pavyzdžiuose.

Tyrimo objektas: Tyrime buvo naudojami seilių ir kraujo serumo pavyzdžiai, surinkti LSMU ligoninės Kauno klinikos Veido ir žandikaulių chirurgijos skyriuje. Tyrime dalyvavo 45 asmenys: 16 - sergančių burnos plokščialąsteline karcinoma ir 29 – asmenys, kurie gydėsi Veido ir žandikaulių chirurgijos skyriuje, tačiau nesirgo burnos plokščialąsteline karcinoma.

Metodai: Interleukino-1 ir interleukino-6 kiekis seilių ir kraujo serumo pavyzdžiuose įvertintas imunofermentiniu Elisa metodu.

Rezultatai: Gauti rezultatai parodė, kad interleukino-1 vidurkinė koncntracija sergančių seilių pavyzdžiuose buvo (1297,9 pg/ml) statistiškai patikimai didesnė nei kontrolinėje grupėje (531,6 pg/ml). Interleukino-1 vidurkinė koncntracija sergančių kraujo serumo pavyzdžiuose (4,275 pg/ml) taip pat buvo statistiškai patikimai didesnė nei kontrolinėje grupėje (3,45 pg/ml). Interleukino-6 vidurkinė koncntracija sergančių seilių pavyzdžiuose (353,27 pg/ml) buvo statistiškai patikimai didesnė nei kontrolinėje grupėje (160,274 pg/ml). Interleukino-6 vidurkinė koncntracija sergančių kraujo serumo pavyzdžiuose (7,289 pg/ml) nesiskyrė nuo kontrolinės grupės (3,387 pg/ml).

Išvados: Seilių pavyzdžiuose interleukino-1b ir interleukino-6 koncentracija buvo statistiškai patikimai didesnė sergančių burnos plokščialąsteline karcinoma nei kontrolinėje grupėje. Kraujo serume interleukino-1 koncentracija buvo statistiškai patikimai didesnė sergančių burnos plokščialąsteline karcinoma nei kontrolinėje grupėje, tuo tarpu interleukino 6 koncentracija nesiskyrė tarp grupių.

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ACKNOWLEDGMENTS

I would like to express my sincere thanks to LUHS Medical faculty for their financial support of this research project. And I would like to express my special thanks to Dr. Rasa Baniene for herguidance and enormous support during this thesis project, and also my special thanks for colleague Donatas Cirulis for his helping in collecting patient samples for this study.

I pay my deep sense of gratitude to my family for encouraging and motivating me always to the highest peak.

My thanks to family, friends, colleagues and university staff whom this journey would not have a chance to see the success light without their support.

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CONFLICT OF INTEREST:

This thesis has no conflict of interest.

PERMISSION ISSUED BY THE ETHICS COMMITTEE:

This study was approved by the Bioethics Department of Lithuanian University of Health Sciences,the Committee of Bioethics has granted permission Nr. BEC – OF – 04. (see annexes 1)

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ABBREVIATIONS :

1. Premalignant Lesions (PML).

2. Oral Squamous Cell Carcinoma (OSCC).

3. Tumor Suppressors Genes(TSGs).

4. Interleukin-1Beta (IL-1β).

5. Interleukin-6 (IL-6).

6. picogram per mililitre (Pg/ml ).

7. Janus Kinase (JAK).

8. Signal Transducer and Activator of Transcription (STAT).

9. Phosphatidyl Inositol-3 Kinase (PI3K).

10. Mitogen-Activated Protein Kinase (MAPK).

11. Horseradish Peroxidase (HRP).

12. Vascular Endothelial Growth Factor (VEGF).

13. Tumor Growth Facto r(TGF).

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CHAPTER 1. INTRODUCTION

Oral Squamous Cell Carcinoma is the 11th most common cancer in the world, OSCC has a high mortality rate, and survivors have a high rate of suffers which makes OSCC one of the essential health issues [1]. Despite the easy access to the oral cavity for clinical examination OSCC usually diagnosed only in the advanced stages [2]. Unfortunately, in advanced stages of OSCC, the treatment methods become less effective, and the patients have low survival rate.

Many clinical studies are conducted around the world aiming to develop a useful noninvasive diagnostic and prognostic biomarkers for OSCC that can be helpful in early diagnosis of OSCC.

Tumor biomarker may be a molecule secreted by tumor cells or a specific response of the body to the malignant growth [3]. Many tumor markers derived from serum, oral tissue and saliva has been presumed for early diagnosis of OSCC and to predict prognosis in OSCC patients, authors have reported more than 100 salivary biomarkers, many molecules have been detected in saliva of OSCC patients using several techniques, such as tumor necrosis factor (TNFα), P53, interleukins (8,6,1β), cyfra 21.1, matrix metalloproteinase MMP (2,8), Cancer Antigen (CA-125), Ki67[4]. Other studies showed that salivary levels of DUSP1 had been increased in OSCC while other markers are decreased such as Micro RNAs, mirR-125a, and mirR-200a levels are significantly reduced in the saliva of OSCC[5]. However, using these markers is often limited due to its weak sensitivity and specificity and lack of significant scale validation. From the previously reported studies, the marker of interleukin (IL- 6) was found to be an important salivary marker for OSCC [6].

Saliva is a biological fluid composed mainly of water and less than 1% protein, electrolytes and other low molecular weight molecules [7]. The importance of saliva has increased in the last decade, and it has been shown that many salivary particles can be used for the diagnosis of oral cancer or other systemic diseases. Saliva is an essential fluid for tumor biomarkers detection, and it provides a simple non-invasive diagnostic method for disease detection [8]. Many studies have shown that salivary biomarkers are useful in detection of lung cancer, breast cancer, gastric cancer, pancreatic cancer [9].

Saliva is secreted from three major salivary glands (parotid, submandibular and sublingual),these glands take up several molecules and water from blood to form the saliva, thus most of the blood molecules such as DNAs, RNAs, proteins, metabolites, are found in saliva (mirror of the body) [10, 11].

Till this date the clinical examination and histopathological biopsy have remained the golden standard for OSCC diagnosis, the histopathological biopsy is usually done only to confirm the diagnosis after

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10 clinical examination of the oral mucosa has suspected OSCC, and this test is only informative when visible microscopic changes appear on tumor cells. To increase survival rate and decrease mortality of OSCC we need proper diagnostic and prognostic tumor marker that will help in early diagnosis and treatment of OSCC.

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CHAPTER 2. AIM AND OBJECTIVES

Aim: to analyze and compare the changes in Interleukin-1 and Interleukin-6 concentrations in saliva and blood serum samples of oral squamous cell carcinoma and control group patients.

Objectives:

1. To investigate an compare the levels of Interleukin-1β in saliva and blood serum of oral squamous cell carcinoma patients and control group patients.

2. To investigate and compare the levels Interleukin-6 in saliva and serum of oral squamous cell carcinoma patients and control group patients.

3. To make a statistical analysis of the results.

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CHAPTER 3. LITERATURE REVIEW

3.1.Oral cancer

3.1.1.Oral Cancer Epidemiology

Oral squamous cell carcinoma is the most common oral neoplasm, OSCC represents about 90% of all oral malignant tumors. OSCC accounts for 2-3% of all cancers in the world and it is the 11th most common cancer worldwide [12].Themost commonage of presentation is about the fifth decade. In most countries around the world and especially in Europe the incidence of OSCC in the male is higher than in female, this due to more involvement of risky habits and behaviors by men like smoking and chewing tobacco. The ratio of male to female incidence of OSCC has been decreased in the last decade into 1.5:1 [13].Worldwide OSCC age-adjusted mortality rate is around 3-4 per 100,000 for men and 1- 2 per 100,000 for women [14]. The survival rate of OSCC is low, and it has not improved in the last decade despite the development of treatment and diagnostic methods, the five-year survival rate has been remaining in the level between 40% and 50% for all stages OSCC with no gender difference of survival rate.The survival rate of OSCC extremely differs between different stages of OSCC. The prognosis and survival rate of OSCC can differ between patients due to several factors; these factors can be divided into three main groups; tumor-related factors, patient-related factors, and treatment- related factors.The tumor-related factors are, anatomical location, TNM staging, histopathological grade, tumor size, tumor invasion, metastasis to lymph nodes, distant metastasis, and molecular markers.Patient-relatedfactors are age, gender, ethnicity, habit, diet, and nutrition.The treatment- related factors are, resection margin, sentinel node dissection, neck dissections, chemotherapy, radiotherapy, patient adherence to cancer treatment [15].

Various studies showed that the anatomical location of OSCC have essential effects on survival rate, for OSCC in the lip or tongue five-year survival rate was around 70% while it wasfor OSCC in buccal mucosa around 30% and for OSCC in maxillary gingiva around 45%.Studies have shown a significant role of TNM staging in the prognosis of OSCC, and besides, to TNM staging other new staging system has been studied by Lee et al. which has shown more accuracy in determining the survival rate of OSCC [16]. However, the prognosis of OSCC is the best when the primary tumor is small and no regional lymph node involvement ordistant metastasis. The survival rate of early-stage OSCC is around 80-90%, while for advanced stages it is about 40% [17].

3.1.2.Anatomical Location

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13 Oral squamous cell carcinoma can develop in differentanatomical locations in the oral cavity, pharynx, or salivary glands. In the Western world, the most prevalent location OSCC are tongue 20-40% of cases and the floor of mouth 15-20% of cases; thus together these two sites account for approximately 50% all cases of OSCC [18, 19]. The tongue and floor of the mouth are common sites of OSCC because they are lined by thin non-keratinized epithelium also due to that alcohol and tobacco smoking substances accumulate in the floor of the mouth and around the base of the tongue. The gingiva, hard and soft palate, retromolar area, buccal mucosa, and labial mucosa are other sites of OSCC, but in the Western world, these sites are less common than tongue and mouth floor[20].In Asian countries OSCC is more common in buccal mucosa 50% of cases,this is due to bettel liquid chewing habits which is verycommon in that countries [21](Figure 1).

Figure 1. Approximate anatomical distribution of oral cancer. This image was taken from [22]

3.1.3. Etiology andRisk Factors:

75% of OSCC cases are associated with modified habits such as tobacco smoking and excessive alcohol consumption; other risk factors include inadequate oral hygiene, inadequate nutrition, poor teeth health, chronic bacterial viral or fungal infections [23]. Many epidemiological studies have investigated risk factors of OSCC, tobacco, and alcohol are the most significant risk factors [24], betel plant chewing is a considerable risk factor in many Asian countries where chewing betel is a common practice, the combination of betel chewing and tobacco have potential risk factor [25], other risk factors include bad oral hygiene and poor dental health [26], infection of human papillomavirus especially type16 is another important risk factor [27]. Despite the critical role of DNA damage and mutations in OSCC development, there is no well-defined hereditary risk factor for oral squamous cell carcinoma.

3.1.5.Oral Cancer Diagnosis

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14 Despite the easy access of oral cavity for clinical examination, OSCC usually diagnosed only in the advanced stages, most common reasons of that delayed diagnosis are the wrong initial diagnosis by the physician and the disregard from the patient and the lack of efficient screening program for OSCC.

Early OSCC often got ignored by the patient because it is asymptomatic[28]. Usually, the patient only asks for medical consultation when the lesion become symptomatic in the advanced stages of OSCC;

symptoms may vary from mild discomfort to severe pain, symptoms may include an earache, bleeding, mobility of teeth, breathing and speech problems, dysphagia, trismus, and paraesthesia [29]. It is essential to establish an early diagnosis in OSCC. OSCC must be suspected in patients with single non-healing oral lesion persisting for more than three weeks. The clinical presentation of early stages OSCC may have different clinical forms (Figures 2, 3) it may be similar to leukoplakia, erythroplakia, or erythroleukoplakia, the most common from is erythroleukoplastic lesion (it consist of slightly rough red and white areas). Any of these lesions of OSCC may develop rapidly into a necrotic ulcer with irregular indurated borders, OSCC lesions may bleed after trauma and could become infected secondarily [30].

Figure 2. Erythroplastic lesion, a carcinoma, on the lateral border of the tongue. Figure was taken from [31].

Figure 3. Leukoplastic lesion , carcinoma, on the lateral surface of the tongue.Figure was taken from [31].

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15 It is very common in the early stages to make the wrong diagnosis, the diagnosis of OSCC is usually delayed up to 6 months. The clinical differentiation between premalignant lesions (PML) in the oral cavity and the OSCC is quite difficult due to the similarity in their signs, symptoms, and on imaging techniques presentation. The biopsy and histopathological examination are quite needed to confirm the diagnosis because the clinical characteristics alone are insufficient [32]. In advanced stages of OSCC, there are classic features of oral malignancy including ulceration, nodularity and fixation to underlying tissues [33].

However, histopathological examinations are expensive procedures and require laboratories with skilled pathologists and lasting long time, and these examinations are only informative when the microbiological changes appear on tumor cells. Patients monitored under screening programs will not be able to undergo biopsies for histological diagnosis; thus histopathological studies are not suitable for routine screening and monitoring. Hence, an appropriate OSCC tumor marker which will be simple and cost-effective method is quite needed for the prognosis and early diagnosis and differential diagnosis of oral PML and OSCC. Also developing proper OSCC tumor marker will help in initiating an effective screening for oral cancer. The suffering, disfigurement, and death associated with oral cancers is definitely avoidable if the OSCC was diagnosed at early stages.

Despite advances in surgery, radiation and chemotherapy, the oral cancer 5-year survival rate has not significantly improved over recent years and remains at approximately 50–55% [34-36].

The mortality rate of OSCC can be reduced through efforts toward developing an early diagnostic method for early diagnosis and screening of high-risk group. Therefore, availability of sensitive and specific biomarkers of OSCC would be highly beneficial [37].

3.1.6.The clinical presentation of OSSC

Sometimes OSCC arises in apparently normal mucosa, but in most cases, OSCC is preceded by premalignant lesions, such as erythroplakia, leukoplakia, erythroleukoplakia and verrucous leukoplakia. Erythroplastic lesions are red plaques, their prevalence is very low between 0.01-0.21%, but erthroplastic lesions have high possibility to develop into severe dysplasia and become malignant lesion, leukoplakia have higher prevalence than erythroplakia, but most of these lesions are not premalignant lesions, homogenous leukoplakia are quite rarely to be premalignant, in contrast, verrucous leukoplakia are more likely to be premalignant. Which, in at least 90% of cases, show severe dysplasia or frank malignancy. In contrast, most white lesions are not malignant or premalignant. OSCC can appear in several forms. OSCC can arise as erythroplakia(red lesion),

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16 leukoplakia(white lesion), erythroleukoplakia, an indurated ulcer, granular ulcer with fissuring or raised exophytic margins, or as a lesion fixed to the overlying skin or mucosa or deeper tissues [38].

OSCC in the advanced stages could spread to several tissues, and most frequent location of distant metastasis is lungs [39].

3.1.7. TNM Clinical Staging of OSCC

OSCC is divided according to the TNM classification into four stages. Stage I: the cancer is 2 cm or smaller, and it's not extending into nearby tissues (T1), the tumor has not spread to adjacent lymph node (N0), or to distant locations (M0). Stage II: the tumor is bigger than 2 cm but not bigger than 4 cm and is not extending into nearby tissues (T2), the tumor has not spread to adjacent lymph nodes (N0), or to distant locations (M0). Stage III : the tumor is bigger than 4 cm (T3), it has not spread to nearby lymph nodes (N0), or to distant locations (M0). Stage IV a: the tumor is any size, and it is extending into nearby structures such as the bones of the jaw or face or deep muscle of the tongue, skin of the face, or the maxillary sinus (T4a), and one of the following,either it has not spread to nearby lymph nodes (N0), or it has spread to 1 lymph node on the same side as the primary tumor but the tumor has not grown outside of the lymph node and the lymph node is not larger than 3 cm (N1), and the tumor has not spread to distant sites (M0). Stage IV b: the cancer is any size and may have extended into nearby tissues or structures (any T) and any of the following, the tumor has spread to 1 lymph node that is larger than 6 cm but has not grown outside of the lymph node (N3a) or It has spread to 1 lymph node that's larger than 3 cm and has clearly grown outside the lymph node (N3b) or It has spread to more than 1 lymph node on the same side, the opposite side, or both sides of the primary tumor with growth outside of the lymph node(N3b) or it has spread to 1 lymph node on the opposite side of the primary cancer that is 3 cm or smaller and has extended outside of the lymph node (N3b). It has not spread to distant organs (M0). Stage IV c: the tumor is any size and may have grown into nearby soft tissues or structures (any T) and it might or might not have spread to adjacent lymph nodes (any N). It has spread to distant sites such as the lungs(M1) [40].

3.2. Oral cancerbiomarkers

Many clinical studies are conducted worldwide aiming to the establishment of a simple and noninvasive diagnostic technique that can differentiate malignant and benign tumors quickly and cost- effectively. Many of tumor markers derived from serum, oral tissue and saliva have been presumed for early diagnosis of OSCC and to predict prognosis in OSCC patients. However, using these markers is often limited due to its weak sensitivity and specificity and lack of significant scale validation. From

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17 the previously reported studies, the marker of interleukin-6 (IL-6) was found to be an important salivary marker for OSCC [41]. IL-6 is the most sensitive marker in early stages of gastric cancer, and it has been suggested as an additional marker in oral cancer. Other studies showed that IL-6 had been produced by tumor cells in esophageal squamous cell carcinoma, multiple myeloma and lung carcinoma [42]. Many studies reported elevated IL-6 levels in saliva of OSCC patients. Studies reveal higher IL-6 levels in serum and saliva of OSCC when compared with premalignant lesions. Also, several studies have reported increased levels of Interleukin-1β in saliva and serum of OSCC patients [43]. Thus in this study, we are aiming to evaluate the level of IL-6 and IL-1β in OSCC patients.

Despite the vast number of research studies supporting the use of IL-6 as a biomarker, there is no validation of this marker in differential diagnosis of OSCC and PMD.

3.2.1. Interleukin-6 signaling in Human Cancer

Interleukin-6 is a multifunctional cytokine that regulates inflammatory responses and many cellular events, IL-6 have several effects on cellular proliferation, migration, invasion, apoptosis, and angiogenesis of tumor cells [44,45]. IL-6 activates the Janus Kinase/Signal Transducer and Activator of Transcription(JAK/STAT) pathway, Mitogen-Activated Protein Kinase(MAPK) pathway and phosphatidylinositol-3 kinase (PI3K) pathway (Figure 4) [45,46]. The IL-6 receptor is composed of two subunits, the ligand binding chain(IL-6R) and the gp130 complex the later one is the responsible for the downstream signal spread [47].

IL-6 binds to IL-6 R which is expressed in several stromal and epithelial cells when IL-6 bind to its receptor it leads to phosphorylation of Janus Kinases (JAK) and phosphorylation of Signal Transducer and Activator of Transcription (STAT) factors, STAT3 is the most important member of STAT family. Activated STAT factors bind to promotor regulatory sequences of targeted genes, and modulate their transcription. Increased STAT3 phosphorylation is considered in several tumors (breast, prostate and hepatocellular cancer) as a marker of severe malignancy[48,49].

Activated STAT3 regulate cellular proliferation and invasion, activated STAT3 also control inflammation, invasion, metastasis, and angiogenesis. Activated STAT3 induces pro-survival and pro-proliferative signaling and contributes to tumor growth of cancer cells [50].

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18 Figure 4. IL-6 binds to its receptor and activate several pathways which lead to tumor growth. Figure taken from [51].

3.2.2. IL-6 Expression in OSCC

Several studies have reported many cytokines are effective in the progression of OSCC, IL-6 is one of these factors which contribute to the development of this tumor. The IL-6 effect in OSCC is similar to its impact on other human cancers, IL-6 bind to its receptor and activate JAK/STAT3, PI3K, MAPK pathways which lead to several effects as it is mentioned above. IL-6 is a bad prognostic factor in OSCC; its higher levels are associated with low survival rate, high rate of relapsing, and metastasis[52]. One of the important IL-6 functions in OSCC is regulation of angiogenesis and lymphogenesis; this effect can be explained by the regulation of vascular endothelial growth factor C synthesis, IL-6 induce vascular endothelial factor C by increasing phosphorylation of Akt [53].

3.2.3. Interleukin-1βsignaling in human cancer

IL-1β is a pro-inflammatory cytokine that is secreted during infection and cellular injury. This cytokine acts directly on several cells, either alone or combined with other inflammatory cytokines. IL- 1β is an inflammatory cytokine that is released when multi-protein innate immune complex called inflammasome (Figure 5) is activated [54]. Inflammasomes has been associated with carcinogenesis

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19 and tumor growth[55]. The inflammasome is a multi-complex system it can be activated by exposure to various carcinogens or by intrinsic genetic mechanisms. Following inflammasome activation, caspase 1 become activated, which cause the cascade of inflammatory events via activation of cytokines, such as IL-1β, then IL-1β interact with their receptors. In addition to that active capsase1 can cause cell pyroptosis with membrane rupture and release of more IL-1β. The inflammasome is also associated with tumor progression through the release of growth factors, such as fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF), that facilitate tumor invasiveness and angiogenesis [54, 55].

IL-1β is a potent marker of carcinogenesis. IL-1β has been identified as a salivary biomarker of OSCC [59]. Also, IL-1β has been reported to be elevated in several cancers, such as breast, skin and melanoma cancers. In melanoma cancer it has been found that levels of IL-1β, IL-6, and IL-8 are elevated; indeed all these cytokines can be regulated by IL-1β [56]. IL-1β have an important role in breast cancer development, a strong association between breast cancer aggressiveness and IL-1β levels have been found, it has been reported that IL-1β levels have been elevated in 90% of invasive breast carcinoma cases [57]. IL1-β producing tumors is considered to have bad prognosis. IL-1 cytokines are a family of cytokines that can induce several genes that promote cancer growth and metastasis, like vascular endothelial growth factor (VEGF) and tumor growth factor (TGF) [58]. IL-1β is found to be the most clinically important subtype of IL-1β family and has got a lot of interest in the last years. For the IL-1β producing tumors, IL-1β must be proteolytically activated by the inflammasome before it can exert its biological effects. IL-1β can enhance the expression of adhesion molecules, increase chemokine release and prostaglandin production. All these factors lead to angiogenesis, chemotaxis, and increase in cell adhesiveness which lead to tumor growth and spread [58]. Many studies have shown that IL-1β is more expressed in saliva than in blood serum and that supports saliva as a proper biological fluid for detecting changes of IL-1β in OSCC [59].

Figure 5. The role of IL-1β in carcinogenesis.this image was taken from [60].

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CHAPTER 4. RESEARCH METHODOLOGY AND METHODS

This study was approved by the Bioethics Department of Lithuanian University of Health Sciences,the Committee of Bioethics has granted permission Nr. BEC – OF – 04. (see annexes 1 ).

4.1. Control and Experimental group:

The study has involved 45 patients from the Facial and Maxillofacial Surgery Clinics of LSMU KK hospital. Written and informed consents were obtained from all the study participants before drawing blood and collecting saliva. In the OSCC group 16 patients were involved, ages from 39–74 y.o, the mean age of them was 57.81±11.94 y.o, 11 males and 5 females. The control group consisted of 29 of non-OSCC patients, ages from 39-74y.o, the mean age of them was 57.56 y.o, 14 males and 15 females.

The experimental group consisted of oral floor OSCC patients who have no history of other malignancy in the past. The primary OSCC tumors of these patients were diagnosed clinically and confirmed by biopsy results. The samples of blood and saliva were collected from patients before surgical treatment, and patients have not been exposed to chemotherapy or radiotherapy before samples collection. OSCC patients were divided according to the Tumor size, clinical data, regional lymph node metastasis, distant metastasis into 4stages as it is mentioned in the literature. Our experimental group was composed of 0 patients from stage-I, 3 patients from stage-II, 8 patients from stage-III, 5patients from stage-IV. Patients who have other malignant tumors other than OSCC or patients who have received chemotherapy or radiotherapy have been excluded from the experimental group.

The control group consisted of patients who have visited the department of facial and maxillofacial surgery for the treatment of facial bone fractures, fibroma, and hemangioma. We excluded from the control group patients who have a history of facial and maxillofacial malignant tumors and patients who are younger than 40 y.o. and patients who have inflammatory disorders.

4.2. Sample collection:

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21 Saliva and blood samples were collected from the 45 patients who participated in the study.

Unstimulated Saliva samples were collected between 7 and 7:30 AM. Participants were asked to refrain from brushing their teeth, eating, chewing, smoking, or drinking anything after they wake up and to collect their saliva in a sterile screw cap. The participants were asked spit their saliva in the collecting tube once a minute for 15-20 minutes, the volume of collected saliva sample was 4-7 ml.

Saliva samples were collected from OSCC patients before any therapeutic procedure. Following collection, the saliva samples were immediately centrifuged at 6800 rpm for 10 minutes at a temperature of 4^oC to remove cell debris, thus the saliva fluid become separated from the sediment which remained in the bottom of the tube, then salivary fluid is collected and put in 1.5 ml Eppendorf tubes, and we add 1.5 µl of proteinase inhibitor to every tube and label each sample. After preparing the samples, we immediately stored them at -80℃ and kept them frozen until the date of the experiment.

Blood samples: 2 ml of peripheral blood were drawn from all study subjects through venipuncture.

Blood was collected into an empty vacutainer. Following collection, blood samples were sent for centrifugation to separate serum from other cellular components of blood, after centrifugation the blood serum separate from cellular components of blood which remains in the bottom of the tube.

Then we put blood serum in 1.5 ml Eppendorf tubes. After preparing the samples, we immediately stored them at -80℃ and kept them frozen until the date of the experiment.

4.3. Human IL-6 estimationin human samples

Concentrations of salivary and serum IL-6 were investigated byavailable commercially human IL-6 ELISA kit(Invitrogen ). The assay was carried out according to the protocol of the manufacturer. The Kit was based on solid-phase sandwich ELISA method (Figure 6) and the procedure is as follows: all collected saliva samples are thawed. In the precoated IL-6 antibody micro plated wells, one control well was filled with only standard solution which contains well knownstandardIL-6concentration to calibrate the curve of light absorbance and calculate the IL-6 concentration. Samples of experimental and control groups were filled in the wells in two sets, two wells for every sample, one well was filled with 50 µl of non diluted saliva, another well was filled with 50 µl saliva diluted with 50 µl standard solution (by 1:1 concentration), then 50µl of IL-6 biotin conjugate was added to every well and incubted for 2 hours at room temperature, next 100 µl of streptavidin-HRP solution was added into each well and incubated for 30 minutes at room temperature, after incubation all wells were washed with 1x buffer wash 4 times, then 100µl stabilized chromogen (tetramethylbenzidine) was added to each well and incubated for 30 minutes at room temperature in the dark. The substrate solution will

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22 begin to turn to blue. Finally, 100 µl of stop solution was added to each well; thus the solution in the wells start to change from blue to yellow ( see the Figure 7). The intensity of color depends on IL-6 concentration which was measured in a microplate reader (Tecan Sunrise, Austria) at a wavelength of 450nm. The concentration of IL-6 in each sample is calculated from the resulting calibration curve.

The results were expressed as pg/ml of saliva or serum.

Figure 6. Principal ELISA scheme for IL-6 determination in saliva and serum samples

Figure 7. Changing of wells color from blue to yellow after adding stop solution.

4.4. Human IL-1β estimationin human samples

Concentrations of salivary and serum IL-1β were investigated by sandwich EAISA kit method (Thermo Fisher Scientific)(Figure 8). The assay was carried out according to the protocol of the manufacturer and procedure as follow: following the receivement of EASIA kit from the distributor all collected saliva samples are thawed. In the precoated IL-1β antibody micro plated wells, one control well was filled with only standard solution which contains well known standard IL-1β concentration to calibrate the curve of light absorbance and calculate the IL-1β concentration. Samples of experimental

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23 and control groups were filled in the wells in two sets, two wells for every sample, one well was filled with 50 µl of non diluted saliva, another well was filled with 50µl saliva diluted with 50 µl standard solution (by 1:1 concentration), then 50 µl of anti IL-1β HRP conjugate was added to every well and incubated for 2 hours at room temperature on a horizontal shaker set on 700 rpm. After incubation all wells were washed with 1x buffer wash 3 times, then 200 µl stabilized chromogenwas added to each well and incubated for 15 minutes at room temperature in the dark on a horizontal shaker set at 700 rpm, the substrate solution will begin to turn to blue, finally 50 µl of stop solution (H2SO4, 1.8N) were added to each well, thus the solution in the wells start to change from blue to yellow (figure 7)The intensity of color depends on IL-1β concentration which was measured in a microplate reader (Tecan Sunrise, Austria) at a wavelength of 450 nm. The level of IL-1β in each sample is calculated from the resulting calibration curve. The results were expressed as pg/ml of saliva or serum.

Figure 8. Pricipal EASIA scheme for IL-1β determination in saliva and blood serum samples

4.5.Statistical analysis

All statistical analysis were done using Microsoft Excel 17, for Windows 10, and the statistical software program SPSS.

Data were expressed in the form of mean of 2-3 repetitive runs for each sample ± SE. α value was considered 0.05, value of P<0.05 was considered signifigant, the value of P<0.001 was considerd very significant. The statistical confidence interval were calculated for 95% confidence. Student-t test was used to check the significance of the study group observations. Spigogram program was used to create the graphs.

CHAPTER 5. RESULTS

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24 In this study, we investigated the level of IL-1β, IL-6 in saliva and serum of oral floor Squamous Cell Carcinoma patients (16 patients) and compared it with the level of IL-1β and IL-6 in saliva and serum of control group (29 person).

4.1. Analysis of level IL-1β in saliva

We found that the mean IL-1β concentration in saliva of oral squamous cell carcinoma patients was 1297.9 pg /ml (SE 213.3 pg/ml), confidence interval was 1297.9 ± 454.6 pg/ml (Figure 9). In control patients the mean IL-1β concentration in saliva samples was 531.6 pg/ml (SE 101.88 pg/ml), confidence interval was 531.6 ± 210.275pg/ml. We found that mean IL-1β concentration in saliva of OSCC patients are higher than its mean concentration in control group. By student t-test, we found that there is a significant difference between values mean IL-1β concentration in experimental group and control group (P=0.001).

Figure 9. Comparision between mean concentration of IL-1β in saliva of OSCC (n =16) and control group (n = 29).

4.2. Analysis oflevelIL-1β in blood serum:

*

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25 We found that the mean IL-1β concentration in serum of oral squamous cell carcinoma patients was 4.275 pg/ml (SE 0.2 pg/ml), confidence interval was 4.275 ± 0.429 pg/ml (Figure 10). In control patients the mean IL-1β concentration in serum samples was 3.449 pg/ml (SE 0.1239 pg/ml), confidence interval was 3.449 ± 0.255 pg/ml. we found that mean IL-1β concentration in saliva of OSCC patients are higher than its mean concentration in control group. By student t-test, we found that there is a significant difference between values mean IL-1β concentration in experimental group and control group (P=0.005).

Figure 10. Comparision between mean concentration of IL-1β in serum of OSCC (n =16) and control group (n = 29).

4.3. Analysis of level of IL-6 in saliva

We found that the mean IL-6 concentration in saliva of oral squamous cell carcinoma patients was 331.19 pg/ml (SE 51.56 pg/ml), confidence interval was 331.19 ± 109.89pg/ml (Figure 11). In control patients the mean IL-6 concentration in saliva samples was 160.274 pg/ml (SE 37.53 pg/ml), confidence interval was 160.274 ± 82.6 pg/ml. we found that mean IL-6 concentration in saliva of OSCC patients are higher than its mean concentration in control group. By student t-test, we found that there is a significant difference between values mean IL-6 concentration in experimental group and control group (P=0.031).

*

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26 Figure 11. Comparision between mean concentration of IL-6 in saliva of OSCC (n =16) and control group (n = 29)

4.4. Analysis of level of IL-6 in blood serum

We found that the mean IL-6 concentration in serum of oral squamous cell carcinoma patients was 7.289 pg/ml (SE 1.757 pg/ml), confidence interval was 7.289 ± 3.83 pg/ml (Figure 12). In control patients the mean IL-6 concentration in serum samples was 3.387 pg/ml (SE 1.024 pg/ml), confidence interval was 3.387 ± 2.17 pg/ml. we found that mean IL-6 concentration in saliva of OSCC patients are higher than its mean concentration in control group. By student t-test we found that there is no significant difference between values mean IL-6 concentration in experimental group and control group (P=0.065), thus there is high probability that the higher level of mean concentration of IL-6 in serum of samples of OSCC patients does come by chance only, and it is not significantly higher than level of IL-6 in control group.

*

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27

IL-6 concentration in serum, pg/ml

0 2 4 6 8 10

Oral cancer Control

Figure 12. Comparision between mean concentration of IL-6 in serum of OSCC (n =16) and control group (n = 29)

4.5 Analysis of level IL-1β and IL-6 insaliva of different stages of OSCC

We found that Mean concentration of IL-1β in Saliva samples of Stage II OSCC is 689.33 pg/ml (n=4, SE= 122.66 pg/ml) (Figure 13). Mean concentration of IL-1β in Saliva samples of Stage III OSCC is 1539.171 pg/ml (n=8, SE= 365.57 pg/ml), Mean concentration of IL-1β in Saliva of samples of Stage IV OSCC is 1423.93pg/ml (n=4, SE= 321.99pg/ml).

Themean concentration of IL-6 in saliva samples (Figure 13) of Stage II OSCC is 223.89 pg/ml (n=4, SE= 57.47 pg/ml). Mean concentration of IL-6 in saliva samples of Stage III OSCC is 392.944 pg/ml (n = 8, SE = 86.1 pg/ml). Mean concentration of IL-6 in Saliva of samples of Stage IV OSCC is 409.71 pg/ml (n = 4, SE = 80.67 pg/ml).

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IL 1 and IL 6 concentration, pg/ml

0 500 1000 1500 2000

IL 1

IL 6

OSCC stage

II III IV

Figure13. Comparison of IL-1β and IL-6 concentration changes in saliva samples of different stages of OSCC.

CHAPTER 6: RESULTS DISCUSSION.

OSCC is reported to threaten the lives of many people in the worldevery year, several surgical and non-surgical treatment methods have been applied in the control of OSCC, but they have not reduced rates of mortality or relapse.In the recent years, new histological markers and inflammatory mediators are found to be able to detect the progression of OSCC. Several studies supported the fact that multiple biomarkers are released in OSCC [61].Inflammatory conditions in the oral cavity cause increasing in cytokine production which lead to increase serum levels of IL-1β, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α). Increased concentrations of these markers in serum and saliva provides the essential for their use in the prediction of OSCC [62].

In particular, IL-6 is an important factor in detectingmalignant growth of tumor cells.IL-6 level significantly increased in renal cell carcinoma, OSCC and lymphoma [63]. In our study we have detected increased levels of IL-1β and IL-6 in saliva of OSCC compared with control group, while the level of IL-1β in saliva of OSCC patients was higher than in control group, there was no significant difference between serum level of IL-6 in OSCC and control group. Our study supported other studies

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29 which suggested IL-6 and IL-1β as potential salivary markers for OSCC. Our study also supported IL- 1β as a serum biomarker for OSCC while for IL-6 levels in serum shows no difference between OSCC and control group subjects.

A study by Korostof et al. found that, salivary levels of IL-1α, IL-6, IL-8, vascular endothelial growth factor A (VEGF-a) and TNF-α were found to be useful in prediction of OSCC at tongue, serving as potential biomarkers for cancer screening and early detection[64]. In a study by Rhodus et al. an increased salivary concentrations of IL-1,IL-6, and IL-8 was detected in OSCC [65]. Katakura et al.

detected higher salivary IL-1β and IL-6 levels in OSCC patients as compared to healthy subjects [66.].

Saheeb Jamee et al. detected significantly higher salivary IL-6 levels in OSCC patients as compared to healthy subjects [67].

A study by St John et al. have detected increased levels of IL-8 and IL-1β in saliva and serum of OSCC patients [68]. Other study by Liu et al. Which invoveld 32 OSCC patients have dected increased salivary levels of IL-1β in OSCC patients [69]. A study by Arrellano-Garcia et al. have shown a significant increase in saliva concentation of IL-1β of OSCC patients as compared to healthy subjects [70]. In a study by Cheng et al. significant difference between levels of IL-6 in saliva of OSCC patients and oral lichen planus patients has been detected [71]. This finding supported that IL-6 may become potential biomarkers for OSCC. This finding was also supported in another study by Saheb Jame et al. who detected a very high serum levels of IL-6 up to 15.9 pg/ml in OSCC [69].Other studies done by Duffy et al. and Hamad et al. who measured the sensitivity of IL-6 in detecting OSCC they have reported that the sensitivity of IL-6 was 80% in serum and 73% in saliva. They defined IL-6 as a sensitive diagnostic biomarker for OSCC [73].

Other clinical studies have confirmed that IL-6 in serum is higher in OSCC than in healthy patients [74-76]

In a study by Chang et al. reported that OSCC patients with higher pretreatment serum IL-6 concentrations had a worse prognosis [77]. Jablonska et al. has detected significantly higher concentration of serum IL-1β of OSCC as compared to healthy subjects [78].

Despite the controversies, IL-6 is suggested as a biomarker for early diagnosis and follow-up to detect relapse in oral cavity carcinomas [79]. Since patients are commonly diagnosed with oral cavity carcinoma at the later stages of the disease, it is expected that serum IL-6 levels increase with cancer stage. The increasing inflammation and lesion region would increase the serum IL-6 level.

Masaaki et al. reported the remarkably high serum IL-6 levels in invasive tumors [80]. In his study, a strong connection existed between serum IL-6 levels and excessive weight loss, invasiveness of the tumor to surrounding tissues, lymph node involvement, and impossibility of complete tumor resection.

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30 Similar findings were reported by Riedel et al. between the average serum IL-6 level and the cancer stage [81].

Thus, our study is supported with all the previous studies which i listed and strongly presume IL-1β and IL-6 as potential biomarkers for diagnosis and early predicion of OSCC. Serum and saliva levels of IL-1β and IL-6 in OSCC patients were elevated according to several studies.

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CHAPTER 7. CONCLUSION

1. IL-1β level in saliva and serum of OSCC was significantly higher than in control group.

2. IL-6 level in saliva of OSCC was significantly higher as compared to control group, while there was no significant difference between IL-6 level in serum of OSCC and control group

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CHAPTER 8. PRACTICAL RECOMMENDATIONS

The results of our study showed that further studies are recommended to accept IL-1β and IL-6 as potential markers for diagnosis or predicting OSCC and for evaluation of prognosis of OSCC.

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KEYWORDS.

1. Oral Squamous Cell Carcinoma.

2. Interleukin-6.

3. Interleukin-1β.

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CONFLICT OF INTEREST:

TABLE OF CONTENTS

SUMMARY

SANTRAUKA

ACKNOWLEDGMENTS

CONFLICT OF INTEREST:

PERMISSION ISSUED BY THE ETHICS COMMITTEE:

ABBREVIATIONS :

CHAPTER 1. INTRODUCTION

CHAPTER 2. AIM AND OBJECTIVES

CHAPTER 3. LITERATURE REVIEW

CHAPTER 4. RESEARCH METHODOLOGY AND METHODS

CHAPTER 5. RESULTS

*

*

*

CHAPTER 6: RESULTS DISCUSSION.

CHAPTER 7. CONCLUSION

CHAPTER 8. PRACTICAL RECOMMENDATIONS

KEYWORDS.

CHAPTER 9. REFERENCES

ANNEXES :

Annex 1