Glioblastoma, also known as di?use astrocytoma or glioblastoma multiforme (GBM), WHO grade IV consists a fatal disease, is considered as the most aggressive and frequent primary brain cancer characterized by severe symptoms and clinical signs that affects the patients’ life quality and extremely poor prognosis and shows a great morphological and genetical heterogeneity. GBM’s incidence is about 5-6 cases/100,000 population and its frequency varies between 12.0%-15.0% of all intracranial tumors. GBM prognosis is extremely poor as the five year survival is only 5.0%, whereas its treatment consists of extensive surgical resection, radiotherapy, and con comitant and adjuvant chemotherapy with temozolomide, however the median survival is short, only about 12 to 15 months after the final diagnosis [1-2]. The main reasons for the poor pro-gnosis is that GBM is an extremely invasive and proliferative tumor with highly abnormal vascularization, displays resistant to the common chemotherapy and radiotherapy and in general is difficult to be completely removed surgically [3]. GBM can be presented as a primary or as a secondary tumor as a result of a malignant trans-formation from a lower grade brain tumor and/or with mutation in the gene of isocitrate de-hydrogenase (IDH) and is classified into several histopathological types such as Classical, Proneural, Neural and Mesenchymal according to the gene expression profile [4-6]. GBM’setiology still remains unclear. Genetic influences in combination with environmental risk factors have been suggested as pathogenic factors of GBM [7]. It has also been suggested that is related with genetic diseases such as tuberous sclerosis, Turcot syndrome, multiple endocrine neoplasia type IIA and neurofibromatosis type I, NF1 [8-11]. In addition, acquired head traumas, which occurred as a result of a brain contusion, may pre-dispose to the development of GBM [12-13]. Among females, a higher risk of GBM has been found in postmenopausal women, and an hypothesis was suggested regarding the possible role of gender hormones in its onset [14]. High levels of height and BMI (Body Mass Index) are also associated with an increasing risk of GBM development [15]. Infections with viruses, such as human cytomegalovirus (CMV) ionizing radiation, chemical agents such as polycyclic aromatic hydrocarbons (PAH), electromagnetic fields and certain metals are also considered to be as risk factors for GBM development [16-19]. Periodontal disease (PD), gingivitis and especially periodontitis is the most common destructive and progressive disease of one or more periodontal tissue structures worldwide and its presence may have systemic effects in several organs such as heart, lungs, etc., as several researches have observed significant associations between PD and systemic diseases and disorders [20]. To be more specific significant associations have been found between PD and cardiovascular disease, atherosclerotic disease, diabetes mellitus, respiratory diseases such as COPD, cancer, etc [21]. The possible causative role of PD in cancer development has been investigated by several authors in different organs such as oral cavity, oesophagus, lungs, stomach, pancreas with conflicting results, even after controlling for potential confounders such as smoking status, socio-economic level, etc. In contrast to the mentioned researches, few studies have investigated the oral conditions or periodontal status in patients who suffered from GBM or other types of cancer, such as gastric and lung cancer [22-30]. The present research was performed to assess the possible differences in periodontal condition between individuals who suffered from GBMdiagnosed by histological examinations and healthy ones.

Study sample

The study sample included 227 individuals, 133 males and 94 females aged 45-77 years which were selected from a Neurosurgery Clinic and two private practices, a medical and a dental one between February 2017 and June 2019. Participants included in the study filled in a medical and a dental health questionnaire and were examined clinically regarding their periodontal status.

Patient’s selection criteria

77 GBM patients referred by the mentioned Clinic, private medical and dental practice accept the invitation to participate in the study. The diagnosis of GBM was confirmed by the histopathological examination after the surgical removal of the tumor. 150 healthy individuals who were referred by a dental practice determined the control group. Both groups, cases and controls, were selected from the same city population in order to avoid or eliminate possible selection biases which can lead to biased associations. To avoid such out-comes a statistical approach for confounding control is the selection of non-cancer individuals to be based on GBM patients’ environment, such as friends, colleagues, etc. To be more specific cases and controls were matched regarding their gender, age, and smoking status (current/previous smokers and never smokers). Gender age and smoking history have been found to be as the principle risk factors for periodontitis development as covariates according to epidemiological studies thus, both groups were matched for the mentioned parameters. For each GBM patient, two healthy individuals with the same gender and of the same age (± 4 years) were selected [31-34]. For four of the 77 cases, no controls were found to match the mentioned criteria. Cases and controls should meet the criteria of established periodontitis and should have atleast a mean of 20 natural teeth, since less than 20 natural teeth could affect the evaluation of the clinical indices examined. Cases and controls ought also not to have been treated by any type of periodontal treatment, conservative or surgical, during the previous six months or prescription of antibiotics or anti-inflammatory or other systemic drugs during the previous six weeks [35-36]. In the study were not included diabetes mellitus patients, patients with cardiovascular diseases, rheumatoid arthritis, liver cirrhosis, immuno-suppressed patients or those who received treatment for the mentioned diseases, and those who received glucocorticoids, in an effort to avoid as much as possible, potential confounding influences on the indices examined. Individuals with brain metastases due to a different primary location, were also excluded from the study design and protocol. The mentioned conditions /disorders could have potential effects on the oral tissues. No oral hygiene instruction was given to the patients’ group for a period of two weeks after the final diagnosis of GBM and before the application of any medical treatment, i.e. surgery, radio-therapy or chemotherapy, as a major problem would have been occurred regarding the intra-examiner variance.

Oral clinical examination

The dental records concerned the following variables, gingival index(GI), PPD and CAL and included all permanent teeth, except 3^rdmolars and the remaining roots using a Williams 12 PCP probe (PCP10-SE, Hu-Friedy Mfg. Co. Inc., Chicago, IL, USA) at six sites per tooth (disto-facial, facial, mesio-facial, disto-lingual, lingual and mesio-lingual).

Gingival Index (GI)

The presence or absence of gingival inflammation was determined by the examination of six sites per tooth. Gingivitis severity was coded as follows:-score 0: normal situation of gingival tissue and/or mild gingival inflammation that corresponds to Löe and Silness classification as score 0 and 1, respectively, and -score 1: moderate/severe gingival inflammation that corresponds to Löe and Silness classification as score 2 and 3, respectively [37].

Periodontal Tissue Examination

The evaluation of PPD and CAL based also on the examination of six sites per tooth. In cases the Cement-Enamel Junction (CEJ) was covered by a restoration or the tooth cervix was destructed by abrasion, decay or another lesion the CEJ location was recorded by extrapolating the CEJ location from the adjacent teeth. In case the CEJ location was not visible, no record was assessed. PPD was coded as follows: score 0: moderate periodontal pockets, 4-6.0 mm and-score 1: advanced periodontal pockets, >6.0 mm. CAL severity was coded as follows score 0: mild,1-2.0 mm of attachment loss, and-score 1: moderate/severe, ≥ 3.0mm of attachment loss. PPD and CAL records concerned the immediate full millimetre [38-39].

Questionnaire

A self-administered questionnaire was filled in by cases and controls and included the following epidemiological variables: age, gender, smoking status, socioeconomic and educational level and information regarding the participants’ medical history with reference to the mentioned systemic conditions/disorders, and medication. A randomly selected sample that was consisted of 35 (20%) individuals was examined clinically by the same dentist after 2 weeks in order to assess the intra-examiner variance. After consideration of the ID’s of the double examined individuals no differences were observed between both clinical examinations (Cohen's Kappa= 0.97). The current case-control study was not an experimental one and was not reviewed and approved by authorized committees (Greek Dental Associations, Ministry of Health, etc). However, it was carried out in full accordance with the World Medical Association Declaration of Helsinki. Individuals who agreed to be included to the study design and protocol signed an informed consent form.

The worst values of the indices assessed were recorded and coded as dichotomous variables after the oral clinical examinations of cases and controls. Females, never smokers, individuals with a low socio-economic (income/monthly equivalent to or less than 1,000 €) and low educational (graduated from Elementary/High School) level were coded as 0. Initially, chi-square test was carried out to estimate the associations between the independent parameters examined and GBM patients and healthy individuals, separately. Secondly, a multi- variate regression analysis model was carried out to examine the associations between GBM as a dependent variable and the independent ones that were determined by the enter method. Adjusted Odds Ratios (OR's) and 95% Confidence Interval (CI) were assessed as well. Finally, the independent variables were included to stepwise method in order to assess gradually the variables that showed significant associations with the dependent one. The statistical method Cohran’s and Mantel-Haenszel’s was carried out in order to control the possible confounders, in an effort to avoid biased secondary associations. Statistical analysis was carried out using the statistical package of SPSS ver.17.0. A p value less than 5% (P< 0.05) was considered to be statistically significant.

Cases and controls showed a mean age of 67.3 years (± 2.88). Univariate analysis is presented in Table 1. No one parameter was found to be statistically significantly different between cases and controls. Table 1 also shows unadjusted OR’s and 95% CI. According to the step 1a of the regression model, the main finding was that CAL and GI were significantly different between patients and controls (Table 2). Table 2 also presents adjusted OR’s with 95% CI and the final step (9a) in which the same indices were significantly different between cases and controls. After using the Cohran’s and Mantel-Haenszel’s method for adjusting known confounders such as smoking and socioeconomic status the same associations were recorded (Table 3).

Table 1: Univariate analysis of cases and controls regarding each independent variable examined. variable examined.

Table 2: Presentation of association between independent variables and GBM cancer according to Enter (first step) and Wald (final step) method of multiple logistic regression analysis model.

Table 3: Application   of   Cohran’s   and   Mantel-Table 3: Application of Cohran’s and Mantel-Haenszel’s, statistical method for controlling possible confounders.

The results showed no statistically significant difference between cases and controls examined, regarding epidemiological indices of gender, age, educational, socioeconomic level, smoking status and PPD, however, OR’s values for socio-economic and smoking status were slightly higher in cases group compared with controls (step 1a) [40-41]. Smoking consists a main causative factor for initiation and progress of PD and various types of cancer and often acts as a confounder in researches that examine the possible association between PD and several types of cancer in which smoking is associated with cancer development. The results showed that smoking was not statistically significant different between cases and controls. PPD is a crucial index for assessing the severity of PD however in the present study that index was not statistically significant different between GBM patients and healthy individuals. In the literature few studies have been performed regarding the oral conditions or periodontal health status in patients who suffered from various types of cancer. In a similar prospective cross-sectional study, individuals with oral or oropharyngeal cancer, showed PPD 6.00 mm or greater of 76.0 % of the patients assessed, whereas only 10.0 % in the control group showed the same severity of disease. The main findings of that study was an association between cancer and more severe PD regardless of oral hygiene and dental health status [42]. A recent case-control study in lung cancer patients showed that PPD was statistically significant different between cases and controls whereas, in another study in gastric cancer patients that finding was not confirmed. The results also revealed that cases showed significantly higher values in gingival inflammation severity, according to GI, compared with controls, finding that cannot be confirmed by previous reports as similar studies have not been performed, except fora recent study which confirmed that finding in gastric cancer patients. On the other hand, the use of that index is limited in epidemiological studies despite the fact that measures the inflammatory load of gingival tissue whereas, Hujoel et al. found that gingival inflammation could be a risk factor for several types of cancer development [24]. As already mentioned a small amount of studies have been carried out regarding the oral conditions in GBM patients or other types of cancer. Critchlow et al [43]. Observed that patients suffered from head and neck cancer had poor oral health at the time of diagnosis, whereas dental caries and PD were suggested as important clinical issues. Another important observation was that GBM patients showed more periodontal problems, i.e. Gingival inflammation and more PD activity in comparison with controls, finding that should be regarded as clinically significant. Those observations regarding the PD indices examined, recorded after controlling potential con-founders such as smoking, and socio-economic status (Table 3). According to the results it is clear that PD appears to be increased in GBM patients and more severe and generalized than in healthy individuals, a clinical sign that concerns all the PD indices examined and would suggest clinical implications for the treatment and management of PD in GBM patients. The higher risk of periodontal tissue lesions in patients with cancer has been suggested to be a result of psychological burden rather than disturbances inpatients’ nutrition or alterations in the oral cavity regarding the quantity/quality of saliva, or disturbances in the balance of micro-biological and immunological parameters in the oral cavity that could be affected because of the chemotherapy or radiotherapy [44-45]. It is also possible that GBM patients are more susceptible to the progression and destruction of periodontal tissue than the healthy population, observation that could be attributed to the extremely poor prognosis of GBM. It has been suggested that the initiation and development of PD and cancer is associated with chronic inflammatory reaction and possible abnormalities in the cellular signaling pathways. Consequently, any type of PD treatment, conservative or surgery could eliminate the levels of biomarkers and mediators that are involved and promote disturbed chronic inflammatory reaction, giving importance to the application of astrictoral care program and preventive dentistry of GBM patients [46]. The purpose of the current research was to investigate a comparison between GBM patients and epidemiologically matched healthy individuals regarding several PD indices and not to investigate a possible relationship between PD indices, as etiological or risk factors, and GBM development. Consequently, the current study has certain limitations that should be taken into account during the procedure of results interpreting. Case-control studies, do not have the reliability of the prospective ones, whereas selection, recall, random biases and the effect of known and un- known confounders are likely higher and could lead to biased secondary associations regarding the variables examined. Another drawback of those studies that are based on questionnaires is that the participants either could not respond or could give no reliable responses, or could over- or underestimate their potential medical diseases or disorders. That results to limitations on the study validity. The decision to be included in the current study older individuals who have at least 20 natural teeth would lead to underestimate those individuals with previous PD who may have had teeth removed for periodontal reasons.

In conclusion, PD as expressed by indices such as CAL and GI was found statistically different in GBM patients compared with non-cancer individuals.

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