Gingivitis and Anti-neutrophil Cytoplasmic Antibodies in children and adolescents suffering from leukemia

VIERA NT, ROJAS DE MORALES T, NAVAS RM, ZAMBRANO OR, PAZ DE GUDIÑO M. GINGIVITIS AND ANTI-NEUTROPHIL CYTOPLAS-MIC ANTIBODIES IN CHILDREN AND ADOLESCENTS SUFFERING FROM LEUKEMIA. MED ORAL PATOL ORAL CIR BUCAL 2004;9:396-402.

ABSTRACT

Introduction: Systemic sicknesses can originate and proliferate alterations in the periodontium. The presence of Anti-neutrophil Cytoplasmic Antibodies (ANCA) in patients suffering destructive periodontal disease and systemic illness has been widely proven.
Objective:To determine the existent relation between gingivitis and ANCA in children and adolescents suffering from leukemia. These patients were examined at the Autonomous Services of the University Hospital in Maracaibo, Venezuela, and at the Hospital of Pediatrics Foundation in Zulia State, Venezuela.
Materials and Methods: 40 patients ranging from 6 to 16 years of age were included in a controlled study. 20 patients suffering from acute leukemia constituted an experimental group, and 20 more patients without apparent systemic sickness who formed a control group. All 40 patients underwent clinical evaluation, and periodontal tissue radiographic evaluation and assessment of ANCA through an enzyme-linked immunoasorbent assay (ELISA); this was done using an IMMCO Diagnostic" commercial kit.
Results: No Significant differences in the gingivitis average index, plaque and bone loss between the experimental and the control group were evidenced. A significant correlation between gingivitis and ANCA was not evidenced either. Nevertheless, 7 out of 20 patients of the experimental group turned out to be ANCA positive. In contrast, no positive cases were reported in the control group. (p<0.05 Fisher Test).
Conclusion: The results of this study suggest that the presence of ANCA is probably related to on going immune alterations in patients suffering from leukemia but not related to gingivitis.

Key words: Gingivitis, ANCA, leukemia.

INTRODUCTION

Gingivitis related to plaque is apparently one of the most common periodontal illnesses and constitutes an inflammatory answer characterized by redness, bleeding and edema of the surrounding gingival tissues as well as loss of the adaptingtissue of the tooth and an increase of the crevicular fluid. The microbial biofilm accumulated in the dental surface is the main etiological factor of periodontal disease. A loss of balance between the microorganisms and the defense mechanisms of the host provokes the development of pathological changes in the periodontium (1). These changes can be aggravated by systemic illnesses and specific disorders interfering with the immune system, having studied leukemia (2,3) congenital neutropenia, (4,5) sarcoma (6) or those associated to non surgical therapies for cancer (7,8).

In patients suffering from leukemia, any organ or tissue can be infiltrated. Thepulp, the periodontium or the oral mucous are not the exception. Frequent oral complications in pediatric patients suffering from cancer are gingivitis (6-9), the gingival enlargement due to the infiltration of leukemic cells, and gingival ulcerations derived from infections with microorganisms of the normal flora in neutropenic patients (2).

On the other hand, the presence of anti-neutrophil cytoplasmic antibodies (ANCA) in patients suffering hematological disorders such as myelodysplasias, acute myeloid leukemia and lymphoproliferative or myeloproliferative syndrome has been widely proven (10).

Anti-neutrophilcytoplasmic antibodies (ANCA) are a type of autoantibody directed against the enzymes located in the primary granules ofmyeloid cells. They were first described in 1982 by Davies et al. (11) in patients with necrotizing glomerulonephritis. Since then, ANCA has been detected in a wide range of inflammatory, infectious, and neoplastic conditions and is currently used as specific marker for Wegener's granulomatosis (12). According to a number of studies, they might be involved in the pathophysiology of disorders such as microscopic polyarteritis, and the idiopathic form of crescent glomerulonephritis (13). Mac Issacet al (14) reported that the presence of ANCA is associatedto the physiology of illness related to vasculitis. Nevertheless, there are not enough evidences that confirm this hypothesis.

Destructive periodontal sickness is frequently associated to systemic conditions in which the neutrophils present some alterations. Novo et al. (15), found ANCA for the first time in patients suffering from periodontitis.

Periodontitis has also been found in patients suffering from erythematosus systemic lupus, thus, suggesting that this illness is the responsible one in most ANCA cases reported from this systemic illness (16).

All of these findings lead to determine the existent relation among gingivitis, the early stage of periodontitis, and the anti-neutrophil cytoplasmic antibodies in children and adolescents suffering from leukemia.

MATERIALS AND METHODS

PATIENTS

A total of 40 children and adolescents ranging from 6 to 16 years of age, all of them residing in Maracaibo, Zulia State, Venezuela, were studied. From those patients, the experimental group was made up of 20 patients who were being treated at the Pediatric Oncology unit of the Autonomous Service at the University Hospital (SAHUM) and at the Pediatrics Specialization Hospital Foundation (FHEP). All of the patients diagnosed with acute leukemia in accordance with the criteria of the French American British (FAB) cooperative working group. Minimal requirement: Having been diagnosed the illness at least six months previously and having neutrophil values of at least > 1500 mm3. Thirteen boys and seven girlsbeing an average age of 10.9 (±3.27) and within a range of 6 to 16 years were included. Concerning the oncological diagnostic, 19 (95%) patients had an acute lymphoblastic leukemia diagnostic, and one (5%) patient had a diagnostic of acute myeloblastic leukemia.

The control group was made up of 20 subjects who had no apparent systemic illness and were selected from the regular dentistry service at the Integral Attention to the Child Center (CIAN), at the dentistry faculty from the University of Zulia,and comprised 10 boys and 10 girls of an average age of 9.85 (±2.32) and ranging from 6 to 14 years of age.

Each one of the parents or representatives of the patients that participated in the study signed their approval and consent after explaining the purpose of the study to them.

EXPERIMENTAL DESIGN

CLINICAL EVALUATION

A clinical historywhich included a periodontal evaluation was carried out in all selected individuals. These evaluations were made early in the morning and included all hospitalized as well as ambulatory patients. All of them were lay down and examined using a halogen frontal light lamp, a flat mouth mirror, and a periodontal WHO probe. The clinical evaluation was made by a trained and calibrated periodontist in a group of 30 children and adolescents. Intraexaminer observation variability for the gingival index (GI) was measured using the Kappa coefficient (0.89).

The GI from Loe and Silness (17), and the Silness and Loeplaque index (PI)(18) were used. The supporting conditionof the dental tissues were evaluated through the depthness of the gingival crevice in six different points of each selected tooth. (Facial: Mesial medial-distal; Lingual: mesial medial-distal), in which the distance from the gingival margin to the location of the tip of the periodontal probe was measured. The bone level was determined through periapical radiographics to evaluate the alveolar crest related to the cement-enamel joint of adjacent teeth. In the cases of primary dentition, teeth 54, 64, 74, 84, 81 and 71 were evaluated, and in the case of permanent dentition, teeth 16, 26, 36, 46, 31, and 41, were evaluated.In mixed dentition cases, in the previous sector, teeth were substituted by the successive permanent ones, and in the posterior sector, the first permanent molars were evaluated.

DETERMINING ANCA

SERUM SAMPLE

Peripheral blood samples from the patients included in this study were obtained through a puncture of the basilic medial vein using the Vacutainer ‚ system without anticoagulant. Six (6) ml of blood were taken from each patient. The samples were kept at room temperature for an hour, centrifugating them at 300g for ten minutes at a temperature of 4ºC . The serum fraction was sucked in using a Pasteur pipette and then was divided into four aliquots at -20ºC until ready to be used. The presence ofANCAwas established throughELISAtest.

ELISA

The presence of ANCA was determined by using ELISA test and an IMMCO Diagnostic" commercial kit. The microplates of Elisa were sensitized using purified proteins such as Myeloperoxidase (MPO) and Proteinase 3 (PR3). The dilution of the samples and the technical procedure was carried out according to the instructions given by the manufacturer of the kit. As a conjugate, anti-human IgG marked with alkaline phosphatase and the specific enzymatic substrate pNPP were used. The readings of the microplates were taken in a 405 nm wave length in an ELISA reader. (Cambridge Tech Inc; Water Town, MA, USA). Sample patients whose values ranged from 20 and 25 EU/ml or more were considered as positive. These values were suggested by the commercial firm.

STATISTICAL ANALYSIS

The differences between gingivitis and plaque indices in the experimental and control groups were determined by the Mann-Whitney test. In the same way, the Fisher exact test was used to establish the differences between the anti- neutrophils cytoplasmic antibodies in the study groups. The correlations between gingivitis andANCA was determined through the Spearman correlation test. A level of probability of 5% was considered statistically significant.

RESULTS

The percentual distribution of PI and GI from the study groups related to the acuteness degree is shown in Table 1. All subject presented some degree of gingivitis, being grade 1 the most frequent. Regarding the plaque index, grade 1 was the predominant value in the control group, whereas in the experimental group was index grade 2.

No significant differences were found related to the GI and PI average value, (Table 2) with Mann-Whitney test p>0.5), between the experimental and control groups. In the subject examined, the gingival groove was between 1 and 2 millimeters depth. No one presented bone loss toradiographic evaluation.

Seven patients (35%) of the experimental group turned out to be ANCA positive. However, the control group did not report any positive cases. These differences were statistically significant. (p<0.05 Fisher exact test). Finally, a significantcorrelation between gingivitis and the presence of ANCA (Spearman r 0.15 p=0.507) was not evidenced.

DISCUSSION

Inthis study, all evaluated patients suffering from lymphohema-topoietic neoplasia presented gingivitis as a type of frequent periodontal illness, coinciding these findings to the ones reported by Fayle & Curson (19), Childers et al (6), and Morales et al (9), who asserted gingivitis as one of the most frequent oral complications in pediatric patients suffering from cancer. However, the prevalenceand acuteness of gingivitis in children and adolescents suffering from leukemia is similar to the one observed in children and adolescentssystemicallyhealthy. Such concept coincides to the reported by Hugoson et al (20), Loe et al (21), Ramberg et al (22) andOh J. et al (23).

As far as the plaque index is concerned, it was evidenced that not only leukemic patients, but systemically healthy ones as well, presented some degree of plaque. The PI did not show any significant difference among the groups studied, contrasting with the findings of Willershausen et al (24) who evidenced significant differences when studying this index in children systemically healthy.

Our results agree with the ones reported by Zambrano et al (25), in a gingival inflammatory response study in children having lymphohematopoietic neoplasia and the ones being systemically healthy. No significant differences betweengingivitis and plaque were found. Besides, GI and PI grade one was the most frequent in both groups.

Onthe other hand, bone loss in the evaluated subjects was not observed in this study, thus coincide to the reported by the Science and Therapy Research Committee of the Periodontal American Academy (1), who pointed out that the destructive periodontal illness is less frequent in children and adolescents compared to adults.

The significant difference observed when associating ANCA levels between the experimental and control groups agrees to the one reported by Savige el al (10), Hamidou et al (26), and Hamidou et al (27), who pointed out the presence of ANCA in patients suffering myelodysplasia and lymphoproliferative syndrome. The presence of these antibodies could be linked to any immune alteration in these patients, as well as the presence of systemic vasculitis. Nevertheless, a significant difference was not found when correlating the presence of gingivitis and ANCA, thus showing that, probably, in the early stage of the periodontal illness, does not exist a positive correlation between them,being different from the one reported by Novo et al.(28), who evidenced significant findings when relating the destructive periodontal illness to the presence of ANCA.

These findings permit to propose, as a hypothesis for future research, that determining ANCA must not be used as an early indicator of destructive periodontal illness since, in the early stages of the same, no tie or bond oftheseantibodies with the gingivitis was observed.

Additional research must be carried out in a greater number of patients to be able to confirm this hypothesis.

ACKNOWLEDGMENTS

To the Scientific and Humanistic Development Council (CONDES) by its financial help; to the Pediatrics Oncology Unit from the Autonomous Service of the University Hospital ofMaracaibo, (SAHUM); to the Pediatric Oncology Unit of the Pediatrics Specialties Hospital Foundation (FHEP); and to the Integral Attention to the Child Center (CIAN) from the Dentistry Facultyof the University of Zulia.

REFERENCES

1. American Academy of Periodontology Committee on Research, Science and therapy. Periodontal diseases of children and adolescents. J Periodontology 1999;70:457-65.        [ Links ]

2. Barret A. Gingival lesions in leukemia: A classification. J Periodontol 1984; 55:585-8.        [ Links ]

3. Genc A, Atayala T, Gedikoglu G, Zulfar B, Kullu S. Leukemic children: Clinical and histoplathological gingival lesions. J Clin Pediatr Dent 1998; 22:253-6.        [ Links ]

4. Goultschin J, Attal U, Golstein M, Boyan B, Schwartz Z. The relationship between peripheral levels of leukocytes and neutrophils and periodontal disease status in a patient with congenital neutropenia. Case repot. J Periodontol 2000; 71:1499-505.        [ Links ]

5. Winkelhoff A, Schouten A, Baart J, Vandenbroucke C. Microbiology of destructive disease in adolescent patients with congenital neutropenia. A report of 3 cases. J Clin Periodontol 2000;27:793-8.        [ Links ]

6. Childers N, Stinnett E, Wheeler P, Wright T, Castleberry P, Dasanayake A. Oral complications in children with cancer. Oral Pathol 1993;75:41-7.        [ Links ]

7. Toth B, Martín J, Fleming T. Oral complications associated with cancer therapy. J Clin Periodontol 1990;17:508 -15.        [ Links ]

8. Peterson D, Dambrosio J. Diagnóstico y terapéutica de complicaciones bucales agudas y crónicas por tratamientos no quirúrgicos del cáncer. Clínicas de Norteamérica 1994;4:451-69.        [ Links ]

9. Morales T, Zambrano O, Rivera L, Tirado D, Fonseca N, Bernardoni C, et al. Oral disease prevention in children with cancer: testing preventive protocol effectiveness. Med Oral 2001;6:326-34.        [ Links ]

10. Savige JA, Chang L, Smith CL, Duggan JC. Anti-neutrophil cytoplasmic antibodies (ANCA) in myelodysplasia and other haematological disorders. Aust N Z J Med 1994;24:282 -7.         [ Links ]

11. Davies D J, Moran J E, Niall J F, Ryan G B. Segmental necrotizing glomerulonephritis with antineutrophil antibody. Br Med J 1982;285:606.        [ Links ]

12. Van der Woude F J, Rasmussen N, Lobattos, Wiik A, Permin H, Van Es L A,et al. Autoantibodies to neutrophil and monocytes: A new tool for diagnosis and a marker in disease activity in Wegeners Granulomatosis. Lancet II 1985;425-9.        [ Links ]

13. Kallenberg C, Brouwer E, Weening J, Cohen J. Antineutrophil cytoplasmic antibodies: current diagnostic and pathophysiological potential. Kidney Int 1994;46:1-15.        [ Links ]

14. Mac Issac A, Moran J, Davies D, Murphy B, Georgiore T, Niall J. Antineutrophil cytoplasm antibody (ANCA) associated vasculitis. Clin Nephrol 1990;34:5-8.        [ Links ]

15. Novo E, García-Mac Gregor, Weir-Medina J, Parra G, Ocando A y Viera N. Anticuerpos anticitoplasmaticos de neutrófilos (ANCA): Un estudio sobre patologías no asociadas con arteritis. Investigación Clínica 1996;37:83-94.        [ Links ]

16. Novo E, García-Mac Gregor E, Nava S, Perini L. A possible defective estimation of antineutrophil cytoplasmic antibodies in systemic lupus erythematosus due to the coexistence of Periodontitis. PRHSJ 1997;16:369 -73.        [ Links ]

17. Loe H; Silness J. Periodontal Disease in pregnancy. Act Odont Scand 1963; 21:533-8.        [ Links ]

18. Silness J, Loe H. Periodontal disease in pregnancy. Acta Odontol Scand 1964;22:121-8.        [ Links ]

19. Fayle S, Curzon M. Oral complications in pediatric oncology patients. Pediatric Dent 1991;13:289-95.        [ Links ]

20. Hugoson A, Koch G, Rylander H. Prevalence and distribution of gingivitis-periodontitis in children and adolescents. Epidemiological data as a base for risk group selection. Swed Dent J 1981;5:91-103.        [ Links ]

21. Loe H, Morrison E. Periodontal health and disease in young people: screening for priority care. International Dental Journal 1986;36:162 -7.        [ Links ]

22. Ramberg PW, Lindhe J, Gaffar A. Plaque and gingivitis in the deciduous and permanent dentition. J Clin Periodontol 1994;21:490-6.        [ Links ]

23. Oh T, Eber R, Wang H. Periodontal disease in child and adolescent. J Clin Periodontol 2002;29:400-10.        [ Links ]

24. Willershausen B, Lenzner K, Hagedorn B, Ernst C. Oral health status of hospitalized children with cancer: a comparative study. Eur J Med Res 1998; 3:480-4.        [ Links ]

25. Zambrano O, Rojas de Morales T, Navas R, Viera N, Tirado D, Rivera L. Respuesta inflamatoria gingival en niños y adolescentes con neoplasias linfohematopoyeticas. Interciencia 2002;27:471-5.        [ Links ]

26. Hamidou MA, El Kouri D, Audrain M, Grolleau JY. Systemic antineutrophil cytoplasmic antibody vasculitis associated with lymphoid neoplasia. Ann Rheum Dis 2001;60:293-5.        [ Links ]

27. Hamidou MA, Boumalassa A, Larroche C, El Kouri D, Bletry O, Grolleau JY. Systemic medium-sized vessel vasculitis associated with chronic myelomonocytic leukemia. Semin Arthritis Rheum 2001;31:119-26.        [ Links ]

28. Novo E, García-Mac Gregor E, Viera N, Chaparro N, Crozzoli Y. Periodon-titis and antineutrophil cytoplasmic antibodies in systemic lupus erythematosus and rheumatoid artritis: a comparative study. J Periodontol 1999;70:185-8.        [ Links ]