Nagham F.doc J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Estimation of 140Pedodontics, Orthodontics and Preventive Dentistry Estimation of some salivary variables and oral health status of patients with chronic myeloid leukemia aged 45-55 years Nagham H. Al-Shaikh Radhi, B.D.S. (1) Athraa M. Al-Waheb, B.D.S, M.Sc. (2) ABSTRACT Background: Chronic myeloid leukemia is a cancer of the white blood cells characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow. This study aimed to determine the effect of chronic myeloid leukemia on Dental caries and Oral health status including Gingivitis, Loss of attachment, Plaque index and Calculus index as well as evaluation of salivary flow rate and salivary interleukins-6 and tumor necrosis factor-α. Material and methods: Study group consisted of (75) subjects, (25) were newly diagnosed with chronic myeloid leukemia, (25) were taking medications (Glevic), and (25) were control subjects, all aged 45-55 years old. Collection of stimulated salivary samples was performed under standard conditions, then salivary flow rate and salivary cytokines estimation was done. Clinical examination and oral health assessment were carried out under the standardized conditions of oral health surveys of World Health organization. dental plaque was scored according to the criteria of plaque Index by Silness and Loe(1964), Dental calculus was scored following the criteria of calculus component of the periodontal Index (Ramfjord, 1959), assessment of gingival health done according to the criteria of gingival Index for Loe and Silness (1963) and loss of attachment done following the criteria of WHO (1997). Caries assessment was done according to the WHO modified Decayed-Missing-Filled Index (DMF) and examination was done with a plane mouth mirror and CPI probe. Results: DMFS index were higher in the newly diagnosed group followed by the control group and then the treated group but the LSD test showed that differences were statistically not significant between each two groups, Plaque index was higher in the newly diagnosed group, and the differences were statistically significant only between the newly and the control group (p<0.05), Calculus index was higher in the newly diagnosed group and the difference was statistically significant between the newly diagnosed group and the treated group. Gingival index was higher in the control group and the difference was statistically highly significant between the new and control groups (p<0.01) and also highly significant between control group and the treated group. Loss of attachment index was higher in the control group the LSD test show that the difference was statistically significant between the control group and each one of the other two groups. In regard to saliva, salivary flow rate was higher in the control group; the difference was significant between the control and the newly diagnosed group and highly significant between control and the treated group. Interleukin-6 level was higher in the newly diagnosed group then the treated group then the control group. LSD test show that the difference was statistically significant between the new and the treated group (p<0.05) and it was highly significant between the new and the control group (p<0.01) but it was not significant between the treated and control group. TNF-α level was higher in newly diagnosed group followed by the treated group then the control group and the difference was statistically significant with the treated group and highly significant with the control group, but the difference was not significant between the treated and the control group. Conclusion: Salivary Interluekine-6 and Tumor necrosis factor- α levels were higher in the newly diagnosed group. Saliva provides an ideal tool for the detection of pro-inflammatory markers. Salivary IL-6and TNF-α may play an important role as biomarkers for CMLand IL-6 is more predictable for the response to the treatment than TNF-α. Key words: chronic myeloid leukemia, myeloid cells, Glevic, salivary cytokines. (J Bagh Coll Dentistry 2013; 25(Special Issue 1):140-145). الخالصة ، فقدان التھاب اللثة بما في ذلكصحة الفم على تسوس األسنان وابیضاض الدم النقیاني تحدید تأثیر :األھداف من الدراسة .ھو سرطان خالیا الدم البیضاء ، یتمیز بزیادة و عدم انتظام نمو أغلب الخالیا النخاعیة في نخاع العظم ، مرض ابیضاض الدم النقیاني:المقدمة ) .ألفا - عامل النخر الورميو 6 - األنترلوكین( العوامل المناعیةمعدل تدفق اللعاب و، و تقییم الترسبات الكلسیة مؤشرو صفیحة الجرثومیةال مؤشر ، ةالتصاق األسنان باللث تم جمع عینات من اللعاب المحفز ، . سنة 55و 45تتراوح أعمارھم ما بین .مشارك سلیم25و،) Glivec( منھم كان یتم عالجھم بالمستحضر 25ابیضاض الدم النقیاني و نھم كانوا مشخصین حدیثا بمرض م 25، ) امشارك 75( شملت الدراسة :المواد و الطرائق و تقییم صحة الفم حسب الظروف القیاسیة الخاصة بمسح ألسریريتم أجراء الفحص كما و .في اللعاب ، و بعدھا تم تقییم معدل تدفق اللعاب و تركیز االنترلوكین )Tenovuo & Lagerlof, 1996( من قبل المذكورة اإلرشاداتحسب الظروف القیاسیة ، بأتباع ,Ramfjord( أمراض ما حول اللثة تم قیاس الترسبات الكلسیة الفمویة حسب مواصفات مؤشر . )Silness and Loe, 1964(ب طریقة حس الصفیحة الجرثومیةتبعا لمواصفات مؤشر الصفیحة الجرثومیةتم قیاس ) . 1997منظمة الصحة العالمیة ( صحة الفم دالة تسوس األسنان (تم تقییم تسوس األسنان حسب مواصفات منظمة الصحة العالمیة ، كما و 1997، كما و تم تقییم فقدان االلتصاق حسب مواصفات منظمة الصحة العالمیة ) Loe and Silness, 1963(تم تقییم صحة اللثة حسب مواصفات مؤشر اللثة . )1959 ) .الدائمیة المعدل . اتبین المجموع كانت الفروقات غیر معنویھو ) 22.20±4.29( المرضى المعالجین وتلیھم مجموعة) 25.96±2.27( الضابطةمجموعة الو تلیھم ) 27.20±5.02(كان أعلى في مجموعة المرضى المشخصین حدیثا مؤشر تسوس سطوح األسنان الدائمیة :ألنتائج كان أعلى في مجموعة فیحات الجرثومیةصشر المؤ . )P< 0.05(الضابطة المرضى المشخصین حدیثا و مجموعة تيو الفروقات كانت ذات داللة إحصائیة بین مجموع ) 1.56±0.11( شر الترسبات الكلسیة كان أعلى في مجموعة المرضى المشخصین حدیثاؤم ة عالیةو الفرو قات كانت ذات داللة إحصائی) 0.49±0.06( الضابطةمجموعة الكان أعلى في التھاب اللثة .المرضى المعالجین المرضى المشخصین حدیثا و مجموعة تيو الفروقات كانت ذات داللة إحصائیة بین مجموع) 0.32±0.06(المرضى المشخصین حدیثا و الفرو قات الضابطةمجموعة المؤشر فقدان االلتصاق كان أعلى في .)P< 0.01( الضابطةمجموعة الو الفروقات كانت ذات داللة إحصائیة عالیة بین مجموعة المرضى المعالجین و ) P< 0.01( الضابطةمجموعة البین مجموعة المرضى المشخصین حدیثا و و الفروقات كانت ذات داللة ) 1.96±0.11(الضابطة مجموعة البخصوص اللعاب ، كان معدل تدفق اللعاب أعلى في .و كل من مجموعتي المرضى المشخصین حدیثا و مجموعة المرضى المعالجین ) 0.56±0.10( الضابطةمجموعة الة بین كانت ذات داللة إحصائی أعلى في مجموعة المرضى المشخصین 6المناعي األنترلیوكین نتركیز البروتیكان . لمجموعة الضابطة ، و الفرو قات كانت ذات داللة إحصائیة عالیة بین مجموعتي المرضى المعالجین و ا الضابطةمجموعة الإحصائیة بین مجموعة المرضى المشخصین حدیثا و الفرو قات و ، ) P < 0.05( الفرو قات كانت ذات داللة إحصائیة بین مجموعة المرضى المشخصین حدیثا و مجموعة المرضى المعالجین و ) 56.0±8.33(ابطة و المجموعة الض) 76.86±18.25(و من ثم مجموعة المرضى المعالجین ) 134.20±60( حدیثا الفا عامل النخر الورمي( تركیز البروتین المناعي .المجموعة الضابطة یر ملحوظة بین كل من مجموعتي المرضى المعالجین و و لكن الفرو قات كانت غ ) P < 0.01(الضابطة مجموعة الكانت ذات داللة إحصائیة عالیة بین مجموعة المرضى المشخصین حدیثا و و مجموعة المرضى المرضى المعالجین ةمجموعبین ذات داللة إحصائیة تفرو قاو كانت ال) 88.60±7.82(و المجموعة الضابطة ) 99.40±6.72(و من ثم مجموعة المرضى المعالجین ) 135.20±13.28( أعلى في مجموعة المرضى المشخصین حدیثا) . المرضى المعالجین ةمجموعة الضابطة و مجموعالبین ةالضابطة ، و لكن الفروقات كانت غیر ملحوظمجموعة ال بین مجموعة المرضى المشخصین حدیثا والفرو قات كانت ذات داللة إحصائیة عالیة و ،) P < 0.05(المشخصین حدیثا بالنسبة لمعدل . المشخصین حدیثا ، بینما ألتھاب اللثة و فقدان االلتصاق كانا أعلى في المجموعة الضابطة الصفیحة الجرثومیة و الترسبات الكلسیة كانت أعلى في مجموعة المرضى. ال توجد فروقات إحصائیة في حالة تسوس األسنان بین المجامیع الثالثة :االستنتاجات ال توجد فروقات معنویھ بین الرجال و . رة ملحوظة في مجموعة المرضى المشخصین حدیثافقد كانا أعلى بصو ) الفا عامل النخر الورمي( البروتین المناعي و 6البروتین المناعي األنترلیوكین تدفق اللعاب فقد كان أعلى في المجموعة الضابطة ، و مستوى تركیز .في مجموعة المشخصین حدیثا والمجموعھ الضابطھ و كذلك مؤشر الصفیحھ الجرثومیھ في المجموعھ المعالجھ) 6- االنترلوكین(النساء في جمیع المتغیرات ما عدا العامل المناعي INTRODUCTION Leukemia is a slowly progressing cancer that starts in blood-forming cells of the bone marrow. (1)M.Sc. Student, Department of Pedodontic and Preventive Dentistry, Dental College, University of Baghdad. (2)Professor, Department of Pedodontic and Preventive Dentistry, Dental College, University of Baghdad. Leukemia cells are the result of an abnormal development of leukocytes (white blood cells) and their precursors. These cells look different than normal cells and do not function properly(1).There are four main types of leukemia, which can be further divided into subtypes. For classifying J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Estimation of 141Pedodontics, Orthodontics and Preventive Dentistry leukemia, the first steps are to determine whether the cancer is lymphocytic or myelogenous, (cancer can occur in either the lymphoid or myeloid white blood cells) and whether it is acute or chronic (rapidly or slowly progressing) (2). Chronic leukemia cells live much longer than normal white blood cells, resulting in an accumulation of too many mature granulocytes or lymphocytes. Chronic leukemia progresses slowly but can develop into an acute form. Major types include chronic lymphocytic leukemia (CLL) and chronic myelocytic leukemia (CML) (3). Chronic myelomonocytic leukemia is a chronic, slowly progressing form characterized by malignant monocytes and myeloblasts, splenomegaly, and thrombocytopenia. Based on clinical characteristics and laboratory findings CML is divided in to three phases: chronic phase, accelerated phase and blast crises phase (4).CML patients usually have a tendency to bleed and high risk of getting infection. Regarding to oral health, a number of scientific studies were carried out to determine the relation between CML and oral health status (dental caries and periodontitis) (5, 6). MATERIAL AND METHODS In this study the sample was consisting of study group (50 patients) with confirmed diagnosis of the disease (CML), 25 of them were newly diagnosed and 25 of them were taking medications which were derivative of 2- phenylaminopyrimidine, (Glevic), for more than one year; and they were examined in Baghdad teaching hospital. In comparison with control group consisting of (25 subject). The samples (both study and control) were aged (45-55 years), (14) males and (11) females. Collection of stimulated salivary samples was performed under standard conditions following the instructions cited by (Tenovuo &Lagerlof) (7). Salivary flow rate was expressed as milliliter per minute (ml\min). The salivary samples were then taken to the laboratory for biochemical analyses. Samples then centrifuged at 4000 rpm for 30 min; the clear supernatant was separated by micropipette and divided into 2 portions, stored at (-20 C) in a deep freeze till being assessed in the laboratory. Clinical examination and oral health assessment were carried out under the standardized conditions of oral health surveys of WHO (8). For dental plaque, selected teeth were examined which was Ramfjord teeth (9).The four surfaces of each tooth were examined and scored following the criteria of plaque Index (PlI) by Silness and Loe (10); this assessment done before saliva sample collection. For Dental calculus, the four surfaces of Ramfjord teeth were examined and scored following the criteria of calculus component of the periodontal Index. Gingival health status, The four surfaces of Ramfjord teeth were examined and scored following the criteria of gingival Index (GI) for Loe and Silness. Loss of attachment (LOA) was done after the Patient's teeth were divided into six sextants, a selected tooth from each sextant was examined by using community periodontal probe (CPI probe), and the maximum score of all the examined teeth were recorded as LOA score of that patient(8). Dental examination was done after collection of salivary sample. According to the WHO modified Decayed-Missing-Filled Index (DMF) Examination was done with a plane mouth mirror and CPI probe. A systematic approach of examination was performed starting from upper right second molar and proceeding in an orderly manner from one tooth or space to the adjacent tooth or space reaching upper left second molar passing to the lower left second molar and then to the lower right second molar(8). Salivary cytokines assessment: (R and D) system, Quantikine Enzyme-Linked Immuno Sorbent Assay (ELISA) (Sandwich technique). Before starting the procedure, all reagents were brought to room temperature and mix gently until the crystals completely dissolved and then the procedure was done according to the manufacturer insetructions for both IL-6 and TNF-α. RESULTS Caries experience: The mean values of DMFS index were higher in the newly diagnosed group followed by the control group and then the treated group but the LSD test showed that differences were statistically not significant between each two groups(p>0.05). Mean value of the decayed surfaces (DS) was higher in the newly diagnosed group. However; the differences were statistically not significant among the three groups (P 0.05).On the contrary the mean value of missing surfaces (MS) was higher in the control group and the differences were statistically not significant among the three groups. .Control group showed a higher value of filled surfaces (FS) and statistically the differences were proved to be significant only between the treated and control groups (P<0.05) (table 1). Plaque index (PlI) was higher in the newly diagnosed group, and ANOVA test show that the difference was significant among the three groups (p<0.05). The LSD test show that the differences J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Estimation of 142Pedodontics, Orthodontics and Preventive Dentistry were statistically significant only between the newly and the control group (P 0.05) (table 2). Calculus index (CalI) was higher in the newly diagnosed group. ANOVA test show that the difference was not significant among the three groups and the LSD test shows that the difference was statistically significant between the newly diagnosed group and the treated group but not significant between the newly diagnosed and the control group (P> 0.05) (table 2). Gingival index (GI) was higher in the control group followed by newly diagnosed group then the treated group. ANOVA test show that the difference was highly significant among the three groups (p<0.01) and the LSD test showed that the difference was statistically highly significant between the new and control groups and also highly significant between control group and the treated group (P<0.01) (table2). Loss of attachment index (LOA) was higher in the control group. ANOVA test show that the difference was not significant among the three groups (p>0.05) but LSD test show that the difference was statistically significant between the control group and each one of the other two groups (p<0.05) (table 2). Salivary flow rate (SFR) was higher in the control group. ANOVA test show that the difference was significant among the three groups, and the LSD test show that the difference was significant between the control and the newly diagnosed group (P<0.05) and highly significant between control and the treated group (p<0.01) (table 2). Cytokines level: Interleukin- 6 levels was higher in the newly diagnosed group. ANOVA test show that the difference among the three groups was significant (p<0.05), LSD test show that the difference was statistically significant between the new and the treated group (P< 0.05) and it was highly significant between the new and the control group (P< 0.01) but it was not significant between the treated and control group (P>0.05) as shown in (table 3). Regarding TNFα, ANOVA test show that the difference was highly significant among the three groups (p<0.01). It s level was higher in newly diagnosed group and the difference was statistically significant with the treated group (P 0.05) and highly significant with the control group, but the difference was not significant between the treated and the control group (P>0.05) (table 3). The Cut off value test: show the sensitivity and the specificity of the ELIZA cytokines test done in this study which was acceptable for both IL-6 and TNF-α since the percent of both is near 70% (table 4). DISCUSSION In this study all participants was found to have dental caries and the difference in DMFS index was statistically not significant among the three groups and this finding was also reported by other studies (5) and it was higher in the newly diagnosed group as those patients had painful mucositis and bleeding tendency leading to alteration in their oral hygiene measures and food type. Saliva may affect caries- experience through its physical and chemical constituent (11).In this study the significant difference in salivary flow rate between the control and the newly diagnosed group may give an explanation for the increase in caries experience in this group, and for the treated group DMFS index was less than the other two groups may be because patients usually give more attention for their oral health to prevent the need for more aggressive dental procedure. Plaque index was higher in the newly diagnosed group with a statistically significant difference with the control group and this result is in consistent with (6) which may explained by the decreased salivary flow rate, neglected hygienic measures and the type of diet of those patients as leukemia cause a burning mucositis that made tooth brushing painful and also made the patients have a tendency toward the soft food (12)In the treated group plaque index become less, this may be because treatment made the patients oral mucosa better so brushing became possible and may because the instructions given for the CML patients regarding the importance of good oral hygiene to prevent the need for any procedure that could cause bleeding or infection (13). Differences in calculus index was significant only between the newly and the treated group, this result can be explained only by the fact that there are many factors effect dental calculus including the amount of dental plaque, which was higher in the newly diagnosed group, salivary flow rate which was lower in the treated group, and salivary composition (14). Gingival index, was higher in the control group with a statistically highly significant difference with both the newly diagnosed and the treated group, and that was a confusing result as plaque index was the least in the control group and many previous studies reported a positive correlation between plaque and gingival indices (15), this result can be explained by the immune disturbances of those patients comparing to the normal subjects that lead to impaired inflammatory response. J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Estimation of 143Pedodontics, Orthodontics and Preventive Dentistry Data of the current study show no impact of the CML on the loss of attachment since it was higher in the control group than in the other two affected groups with a significant difference; this result was expected since gingival index was higher in the control group and can be explained by the difference in the immune factors of the three studied groups. In the newly diagnosed group, IL-6 and TNF-α were highly increased comparing to the control group this may cause decrease in gingivitis and loss of attachment. The mean value of the salivary flow rate was higher in the control group then the newly diagnosed group and the treated group and the differences was significant with the newly diagnosed (p<0.05) and highly significant with the treated group (p<0.01) and the lower value in the newly diagnosed group could be explained by the effect of medications such as analgesics because CML patients usually have abdominal pain before the diagnoses (16). The mean value of IL-6 was higher in the newly diagnosed group than the treated group and the control group and the difference was highly significant with the control (p<0.01) and significant with the treated group (p<0.05), this results could be compared with Anand et al(17) ,who found that the level of IL-6 was significantly higher in the serum of newly diagnosed CML patients because of the fact that saliva is the mirror of the serum (18). This increase in the salivary IL-6 in the newly diagnosed group may explain why gingival index and loss of attachment was higher in the control group comparing to the newly diagnosed one since IL-6 act as pro- inflammatory and anti-inflammatory mediators (19). TNF-α level in the saliva of the newly diagnosed CML patients was the higher with a significant deference with the treated group and highly significant difference with the control group; this result is agreed with the fact that Dysregulation of TNF-α production is involved in many types of cancer and autoimmune diseases (20). Table 1: Comparison of the mean (DMFS) and its component in the three groups enrolled in the present study Table 2: Comparison of mean plaque index, gingival index, calculus index, loss of attachment and salivary flow rate among the three groups. Variables Descriptive statistic Comparative statistic F-value LSD Newly diagnosed group (mean±SE) Treated group (mean±SE) Control group (mean±SE) P1 New + treated P2 New + control P3 Treated + Control PlI 1.56+0.11 1.29+0.11 1.19+0.09 0.034* 0.061 0.012* 0.508 GI 0.71+0.07 0.63+0.06 0.94+0.06 0.002** 0.387 0.009** 0.001** Call 0.32+0.06 0.18+0.03 0.22+0.04 0.085 0.033* 0.108 0.591 LOA 0.28+0.09 0.28+0.09 0.56+0.10 0.062 1.000 0.041* 0.041* SalFR 1.62+0.12 1.47+0.13 1.96+0.11 0.016* 0.383 0.048* 0.005** Variables Descriptive statistic Comparative statistic F- value LSD Newly diagnosed group (mean±SE) Treated group (mean±SE) Control group (mean±SE) P1 New + treated P2 New + control P3 Treated + control DMFS 27.44+5.02 22.20+4.29 25.96+2.72 0.652 0.372 0.800 0.521 DS 11.80+2.79 9.64+3.28 5.20+0.99 0.183 0.551 0.071 0.222 MS 13.76+3.73 11.28+2.60 16.92+2.66 0.426 0.566 0.465 0.194 FS 1.92+0.83 1.28+0.57 3.84+0.90 0.059 0.562 0.085 0.023* J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Estimation of 144Pedodontics, Orthodontics and Preventive Dentistry Table 3: Comparison of mean Interleukin-6 and Tumor necroses factor-α among the three groups Variables Descriptive statistic Comparative statistic F-value LSD Newly diagnosed group(mean±SE) Treated group (mean±SE) Control group (mean±SE) P1 New+ treated P2 New+ control P3 Treated+ control IL6 134.60+25.51 76.86+18.25 56.00+8.33 0.012* 0.033* 0.004** 0.434 TNFα 135.20+13.28 99.40+6.72 88.60+7.82 0.001** 0.011* 0.001** 0.434 Table 4: The Cutoff value for IL6 and TNFα IL-6 Cutoff value = 57.5 TNF Cutoff value = 87.5 Sensitivity 64 % 76 % Specificity 68 % 78 % Area UC 0.730 0.775 P-value 0.005 0.001 Figure 1: Receiver operator characteristic curve (ROC) to determine the cutoff values for both IL6 and TNFα Table 5: Number of positive and negative cases in the newly diagnosed and the treated groups, calculated according to above mentioned cutoff value of IL-6 Negative Positive Groups No. % No. % New 9 36% 16 64% Glivic 16 64% 9 36% Table 6: Number of positive and negative cases in the newly diagnosed and the treated groups, calculated according to above mentioned cutoff value of TNF-α Negative Positive Groups No. % No. % New 6 24% 19 76% Glivic 12 48% 13 52% J Bagh College Dentistry Vol. 25(Special Issue 1), June 2013 Estimation of 145Pedodontics, Orthodontics and Preventive Dentistry REFERENCES 1. 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