Exfoliative Cytology of Oral Mucosa in Kidney Transplant Patients: A Cytomorphometric Study

M. Keles, U. Tozoglu, D. Unal, F. Caglayan, A. Uyanik, H. Emre, K. Cayir, and B. Aydinli ABSTRACT Objectives. The aim of this study was to investigate quantitative cytologic changes in oral mucosal smears collected from kidney transplant patients by modern stereologic methods. Methods. Smears were obtained from the clinically healthy buccal mucosa and oor of mouth of transplant patients (n 21) and healthy volunteers (n 27). Smears from each individual stained by the Papanicolaou method were analyzed using a stereologic method. Nuclear (NV) and cytoplasmic (CV) volumes were evaluated with Stereo Investigator software. Results. CV values were 123,012.71 15,840.96 fL in the oor of the mouth and 133,667.10 26,653.39 fL in the buccal mucosa of the kidney transplant patients. CV values were 133,746.93 14,210.67 fL in the oor of the mouth and 167,797.78 21,007.70 fL in the buccal mucosa of the control group. NV values were 945.68 65.85 fL in the oor of the mouth and 845.15 81.56 fL in the buccal mucosa of the kidney transplant patients and 485.17 18.03 fL in the oor of the mouth and 410.25 52.84 m3 in the buccal mucosa of the control group. Signicantly higher NV (P .000) and NV/CV (P .000) and lower CS (P .000) values were found in the patient group compared with the control group. Conclusion. The ndings suggest that there were alterations in oral epithelial cells, detectable by microscopy and cytomorphometry in kidney transplant patients.
enal transplantation (RT) is an important procedures in the management of patients with end-stage renal disease (ESRD). Some drugs, such as prednisolone, mycophenolate mofetil (MMF), cyclosporine (CsA), and tacrolimus (Tac), are commonly used agents in RT. These drugs have some side effects in the oral region, such as gingival overgrowth, fungal infections, hairy leukoplakia, and increased risk of oral cancer. Early detection of oral cancer in transplant patients is very important owing to mortality and morbidity.1,2 Classic diagnostic methods for precancerous and cancerous oral lesions include clinical examinations and histopathologic studies of biopsied material.3 The most accepted clinical technique for the diagnosis of lesions in the oral mucosa is incisional or excisional biopsy.4 Biopsies of normal and malignant tissues, or scrapings containing exfoliated oral cells, have been explored for many years.5 Exfoliative cytology is a simple nonaggressive technique that is well accepted by a patient and allows a quick and fairly accurate assessment of suspicious lesions of the oral cavity.6 After the cells have been collected, xed, and stained, the morphology of surface epithelial cells is observed under a microscope.7 This technique to examine the morphologic and morphometric aspects of the cell may yet be implemented in public health programs.8 One of the important methods is stereology to make morphometric assessment. A stereology method that is based on fundamental principles of geometry and statistics and is largely concerned with the 3-dimensional interpretation of planar sections of materials or tissues are suggested
From the Department of Nephrology (M.K., A.U.), Department of Histology and Embryology (D.U.), Department of Medical Oncology (K.C.), and Department of General Surgery (B.A.), Faculty of Medicine, Erzurum, Turkey, Department of Oral Diagnosis and Radiology (U.T., F.C.), Faculty of Dentistry, Ataturk University, Erzurum, Turkey; and Department of Nephrology (H.E.), Faculty of Medicine, Yznc Yl University, Van, Turkey. Address reprint requests to Mustafa Keles, MD, Department of Nephrology, Medical Faculty, Atatrk University, 25240 Erzurum, Turkey. E-mail: keles.m@gmail.com 0041-1345/see front matter doi:10.1016/j.transproceed.2011.01.111 871

2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 43, 871 875 (2011)

872

KELES, TOZOGLU, UNAL ET AL The cytomorphometric data were compared between RT and control groups by the independent-samples t test. The statistical analysis was performed using the statistical software package SPSS (version 10.0; SPSS, Chicago, IL, USA). Levels of signicance were set at P .05.

to perform morphometric analyzes. There are some of methods in stereology to obtain the volume parameter of smaller objects, such as cells: optical fractionator with contour trace, Cavalieri estimator, surface-weighted star volume, area fraction fractionator method, and 3- or 2dimensional nucleator.9,10 The aim of the present study was to investigate the quantitative cytologic changes of oral mucosa smears collected from RT patients by modern stereologic methods for 3 reasons. First, we have not found any stereologic study in the literature about morphometric analyses of exfoliative cytology. Second, we sought to examine the methods effectiveness. Finally, we intended to describe the risk of postoperative oral cancer of kidney transplantation patients compared with healthy control subjects.
PATIENTS AND METHODS
A total of 21 patients with renal transplants and 27 healthy volunteers were recruited from the Department of Nephrology, Ataturk University. All procedures followed the tenets of the Declaration of Helsinki. A pro forma inventory was completed detailing name, age, gender, and relevant medical history. Patients with anemia, diabetes mellitus, radiotherapy, alcohol consumption, and smoking were not included in the study. Patients with glomerular ltration rate (GFR) 60 mL/min and 12 months after preemptive RT were included in this study. Smears were obtained from clinically healthy buccal mucosa and oor of the mouth of patients with transplant attending the private clinic and from volunteer control individuals. After clinical examination, the oral mucosa was dried with a gauze swab to remove surface debris and excess saliva, and smears were taken using a cytobrush and transfered to clean dry glass slides. These were then immediately sprayed with a commercial xative containing 95% ethyl alcohol. Smears from each individual stained by the Papanicolaou method were analyzed using the stereologic method of 2 dimensional nucleator. The smears were placed on a motor-driven stage attached to an microscope, and cells were projected onto the monitor via camera at 200 magnication. Each clearly dened cell with predominant staining was examined by systematic sampling in a stepwise manner, moving the microscope stage from left to right and then down and across to avoid measuring the same cells again. The nuclear (NV) and cytoplasmic (CV) volumes were evaluated for each cell using the software Stereo Investigator (Microbrighteld). The volume of interesting cells obtained from the buccal mucosa and oor of the mouth of RT patients and healthy volunteers smears was estimated using the nucleator method11 following the formula: V(N) 4 3 l3 n

RESULTS

In our study, the mean age was 36.10 10.52 years in the RT patients (15 men and 6 women) and 37.30 8.37 years in the control group (19 men and 8 women). There was no difference between the 2 groups regarding age and gender (P .05). The mean time of buccal mucosa and oor of the mouth smears obtainment was 14.9 3.1 months of transplantation. Mean GFR was 82.09 14.54 mL/min and mean serum creatinine 1.12 0.23 mg/dL. Ten patients were using CsA MMF prednisolone and 11 patients Tac MMF prednisolone. No patient had received any monoclonal or polyclonal antibodies. Cytomorphometric results showed that NV in buccal mucosa was markedly higher (P .000) in the RT patient group (845.15 81.56 fL) than in the control group (410.25 52.84 fL), and CV in buccal mucosa was markedly lower (P .000) in the R patient group (133,667.10 26,653.39 fL) than in the control group (167,797.78 21,007.70 fL). In addition, NV in the oor of the mouth was markedly higher (P .000) in the RT patient group (945.68 65.85 fL) than in the control group (485.17 18.03 fL), and CV in the oor of the mouth was markedly lower (P .017) in the RT patient group (123,012.71 15,840.96 fL) than in the control group (133,746.93 14,210.67 fL). Figures 1 and 2. show histologic views of exfoliative cell samples. In this study, we also determined the NV-CV ratio (N/C). These ratios in the oor of the mouth were 0.008 0.001 in RT patients and 0.004 0.000 in the control group. In the buccal mucosa, these ratios were 0.007 0.001 in the RT patients and 0.002 0.000 in the control group. The N/C ratio mean was higher in the RT patient group than in the control group (P .000).
DISCUSSION

where Vn is the number-weighted volume, l is length of intercepts, and n the number of nucleator estimates. Although some requirements for obtaining full randomness in the section-sampling stage while estimating the volume of the small objects by the nucleator method have been mentioned in the literature,1113 we did not implement any requirement associated with section stage of the sampling procedure, because we used a smear.

In this study, we performed microscopic and cytomorphometric analyses of the oral epithelium (buccal mucosa and oor of the mouth) in RT patients, as a result of a lack of previous works regarding this issue. We found that there are quantitative cytologic changes in cells in transplant patients. This may be related to the immunosuppressive agents.14,15 Immunosupressive medication have negative impact on immune surveillance and affect DNA repair mechanisms, leading to irreversible DNA alteration and subsequent carcinogenesis.2 They may cause gingival overgrowth, an increased incidence of Candida spp. in the saliva, and herpes simplex.16 Increased cancer incidence after organ transplantation has been demonstrated in several studies.2,17,18 Villeneuve et al.18 reported that the cancer incidence in RT patients was 2.5 times higher than rates observed in the general

ORAL MUCOSA CHANGES IN KIDNEY TRANSPLANT PATIENTS

873

Fig 1. Histologic view of exfoliative cell samples. Top grid presents buccal mucosa of healthy volunteers and transplant patients. Bottom grid shows exfoliate proles obtained from the oor of the mouth. (A, B, C) Control; (A-1C-3) exfoliate proles of transplant patients.

population. After transplantation, a signicant excess was observed for melanoma, Kaposi sarcoma, non-Hodgkin lymphoma, Hodgkin disease, leukemia, and cancer of the lip, tongue, mouth, salivary gland, esophagus, stomach, colon, anus, liver, gallbladder, lung, connective and other soft tissue, vulva, cervix, penis, eye, thyroid, and unspecied site.2,19 The risk has been especially high in the head and neck sites. Van Leeuwen et al20 suggested that lip cancer risk is associated with the duration of immunosuppression, specic immunosuppressive agents, and ultraviolet light exposure. In our study, the cytomorphometric ndings in the oral smears of transplant patients demonstrated that there was a real increase in the NV, and the CV presented statistically signicant differences. The mean N/C was higher in the RT patient group. These ndings suggest that oral cells may show malignant transformation in RT patients. Ramaesh et al.21 found that cytoplasmic diameter was highest in normal

mucosa, lower in dysplastic lesions, and lowest in squamous cell carcinomas (SCCs) by using cytomorphometric techniques. In contrast, nuclear diameter was lowest in normal mucosa, higher in dysplastic lesions, and highest in SCCs. We think that because increased nuclear size and reduced cytoplasm size may be early indicators of malignant transformation, exfoliative cytology may play an important role for monitoring clinically suspect lesions and for early detection of malignancy in RT patients. Exfoliative oral cytology examines the characteristics of cells that ake off, whether naturally or articially, from the oral mucosa. After the cells have been collected, xed, and stained with Papanicolaou, the morphology of surface epithelial cells is observed under a microscope.7 The basic requirements for a useful technique should be ease of use in any location, minimum trauma, and providing an adequate and representative number of epithelial cells. Cytobrush is an adequate instrument owing to its ease in sampling and

874

KELES, TOZOGLU, UNAL ET AL

Fig 2. Illustration of stereologic estimation according to the nucleator method rst described by Gundersen.14 In this estimation, it was used in Stereo Investigator version 6.

the quality of the oral cytologic sample.7,22,23 Cytobrush penetrated the lesions to ensure that representative material is obtained. The aim is to collect both surface and basal cells, which makes the sample more representative.7 Brush biopsy is a simple, relatively inexpensive, sensitive, risk-free method of screening for cancer and a relatively painless technique that is readily accepted by patients.22 This technique, however, should complement the biopsy, not replace it.3,7 In this study, we showed that there are some quantitative cytologic changes in oral epithelial cells in RT patients that can be detected by quantitative cytomorphometric techniques that are objective, accurate, reproducible, and based on evaluation of parameters such as variations in the size of the NV and CV and alterations in the N/C ratio. It may provide useful information in RT patients to both understand and diagnose some diseases earlier than by other conventional methods.
REFERENCES
1. King GN, Healy CM, Glover MT, et al: Increased prevalence of dysplastic and malignant lip lesions in renal-transplant recipients. N Engl J Med 332:1052, 1995 2. Vasudev B, Hariharan S: Cancer after renal transplantation. Curr Opin Nephrol Hypertens 16:523, 2007 3. Acha A, Ruesga MT, Rodriguez MJ, et al: Applications of the oral scraped (exfoliative) cytology in oral cancer and precancer. Med Oral Patol Oral Cir Bucal 10:95, 2005 4. Jones AC, Pink FE, Sandow PL, et al: The cytobrush plus cell collector in oral cytology. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 77:95, 1994

5. Proia NK, Paszkiewicz GM, Nasca MAS, et al: Smoking and smokeless tobaccoassociated human buccal cell mutations and their association with oral cancera review. Cancer Epidemiol Biomarkers Prev 15:1061, 2006 6. Diniz FM, Garcia GA, Crespo AA, et al: Applications of exfoliative cytology in the diagnosis of oral cancer. Med Oral 9:355, 2004 7. M Prez-Sayns, JM Somoza-Martn, F Barros-Angueira, et al: Exfoliative cytology for diagnosing oral cancer. Biotech Histochem 85:177, 2010 8. Alberti S, Spadella CT, Francischone TRCG, et al: Exfoliative cytology of the oral mucosa in type II diabetic patients: morphology and cytomorphometry. J Oral Pathol Med 32:538, 2003 9. Odaci E, Kaplan S, Sahin B, et al: Effects of low-dose oxcarbazepine administration on developing cerebellum in newborn rat: a stereological study. Neurosci Res Commun 34:28, 2004 10. Aslan H, Altunkaynak BZ, Altunkaynak ME, et al: Effect of a high fat diet on quantitative features of adipocytes in the omentum: an experimental, stereological and ultrastructural study. Obes Surg 16:1526, 2006 11. Gundersen HJ: The nucleator. J Microsc 151:3, 1988 12. Mattfeldt T, Mall G, Gharehbaghi H, et al: Estimation of surface area and length with the orientator. J Microsc 159:301, 1990 13. Nyengaard JR, Gundersen HJG: The isector: a simple and direct method for generating isotropic, uniform random sections from small specimens. J Microsc 165:427, 1992 14. Proctor R, Kumar N, Stein A, et al: Oral and dental aspects of chronic renal failure. J Dent Res 84:199, 2005 15. Cerver AJ, Bagn VJ, Soriano YJ, et al: Dental management in renal failure: patients on dialysis. Med Oral Patol Oral Cir Bucal 13:419, 2008 16. Spolidorio LC, Spolidorio DM, Massucato EM, et al: Oral health in renal transplant recipients administered cyclosporine A or tacrolimus. Oral Dis 12:309, 2006

ORAL MUCOSA CHANGES IN KIDNEY TRANSPLANT PATIENTS 17. Makitea AA, Lundberg M, Salmela K, et al: Head and neck cancer in renal transplant patients in Finland. Acta Otolaryngol 128:1255, 2008 18. Villeneuve PJ, Schaubel SS, Fenton SS, et al: Cancer incidence among Canadian kidney transplant recipients. Am J Transplant 7:941, 2007 19. Vajdic MC, McDonald SP, McCredie M: Cancer incidence before and after kidney transplantation. JAMA 296:2823, 2006 20. van Leeuwen MT, Grulich AE, McDonald SP, et al: Immunosuppression and other risk factors for lip cancer after

875 kidney transplantation. Cancer Epidemiol Biomarkers Prev 18:561, 2009 21. Ramaesh T, Mendis BR, Ratnatunga N, et al: The effect of tobacco smoking and of betel chewing with tobacco on the buccal mucosa: a cytomorphometric analysis. J Oral Pathol 28:385, 1999 22. Mehrotra R, Gupta A, Singh M, et al: Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions. Mol Cancer 5:11, 2006 23. Jones AC, Pink FE, Sandow PL, et al: The cytobrush plus cell collector in oral cytology. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 77:95, 1994