Report

Cytogenetic biomonitoring in oral leukoplakia patients with mild dysplasia Mariano S anchez-Siles1,2, DDS, PhD, Fabio Camacho-Alonso1,2, DDS, PhD, opez-Jornet1,2, DDS, MD, PhD Irene Ros-Llor1,2, DDS, PhD, and Pia L

1 Department of Oral Medicine, University of Murcia, Murcia, Spain, and 2Department of Dentistry, University of Murcia, Murcia, Spain

Correspondence nchez-Siles, DDS, PhD, Mariano Sa Professor of Oral Medicine, University of Murcia Medicina Oral Hospital Morales Meseguer Avda s de los Velez s/n Marque 30008 Murcia Spain E-mail: [email protected]

Abstract Objective A study is made of DNA damage and apoptosis in a group of patients with oral leukoplakia (OL) with mild dysplasia. Materials and methods The study comprised 30 patients with a clinicopathological diagnosis of OL with mild dysplasia and 30 controls. Both samples were similar in terms of age and gender distribution. Brush samples of lesion epithelial cells were collected, followed by cell centrifugation, preparation of the slides, fixation and staining, and analysis under the fluorescent light microscope. The exfoliated cells were examined to detect micronuclei (MN), nuclear buds, binucleated cells, condensed chromatin, pyknosis, and cells with karyorrhexis and karyolysis. Results The patients with OL with mild dysplasia showed a greater frequency of MN (P < 0.001), nuclear buds (P = 0.018), and binucleated cells (P = 0.008). Conclusions Cytogenetic biomonitoring is a simple and scantly invasive technique allowing clinicians to assess DNA damage and apoptosis in patients with OL. Clinical relevance Oral cancer should be detected and controlled in its precancerous stages in order to increase survival rates. Leukoplakia lesions must be biomonitorized periodically. Biomonitorization offers sensibility, no morbidity, speed, and low cost.

Introduction

1454

Oral cancer is the most common head and neck malignancy, with a worldwide incidence of 300,000 cases per year.1 Unfortunately, the 5-year survival rate is less than 50%.2 Patients usually present regional metastases or distant spread at the time of diagnosis – this reflects the existence of a significant delay in detecting the disease.3 Oral leukoplakia (OL) is a premalignant lesion defined by the World Health Organization (WHO) as a white lesion or plaque that cannot be clinically or histologically characterized as corresponding to any other type of disease.4 Oral leukoplakia is present in 60% of all patients diagnosed with oral squamous cell carcinoma (OSCC).5 The detection of malignant and potentially malignant oral lesions in early stages has a favorable impact upon patient survival rate. The risk of malignant transformation of OL with dysplasia is about 43%.6 Oral squamous cell carcinoma is preceded by a series of precursor stages that induce morphological changes in the cells of the oral mucosa, giving rise to clinically detectable potentially malignant lesions. International Journal of Dermatology 2014, 53, 1454–1459

The biological behavior of these lesions is quite unpredictable, as in some cases they may regress or fail to progress, while in one-third of the cases they can progress towards invasive cancer.7 Prediction of the malignant potential of OL on the basis of the pathology findings may be limited. In this context, it would be of great practical importance to develop biomarkers capable of helping in the identification of lesions at high risk of malignant transformation.8 The early diagnosis of oral cancer plays a key role in relation to progression of the disease, the response to treatment, and patient quality of life and survival. Sometimes OL shows no correlation between the clinical and the histological diagnosis. Dysplasia is to be suspected in non-homogeneous leukoplakia, but in some cases OL lesions that clinically have a benign appearance of dysplasia may occur. While dysplasia is associated with a greater risk of malignant transformation, it is also important to control leukoplakia without dysplasia or with mild dysplasia, as in some cases these lesions may transform into carcinoma.9 The oral mucosa constitutes a barrier against potential carcinogens, which in turn may be metabolized to form ª 2014 The International Society of Dermatology

 nchez-Siles et al. Sa

potentially reactive products.10 Up to 90% of all cancers appear to be of epithelial origin. In this context, the oral mucosa could be used to monitor the first genotoxic events resulting from the action of potential carcinogens that come into contact with the body.11 The accumulation of genomic damage leads to genetic instability that can manifest as chromosomal alterations.8 Cytogenetic biomonitoring (CB) is a minimally invasive technique that allows us to evaluate DNA damage, chromosomal instability, and cell death in the oral mucosa. Cytogenetic biomonitoring can be used to detect early genotoxicity events in patients with OL. The technique is rapid, sensitive, inexpensive, and widely used as a reliable tool in patients at high risk of developing cancer.12 The present study explores DNA damage and cell death in a group of patients with OL, compared with a healthy control group. Materials and methods This case–control study was carried out in a total of 60 Caucasian individuals: 30 patients with a clinicopathological diagnosis of OL with mild dysplasia; and 30 controls. The patients were diagnosed and included in the study on a consecutive basis. Both groups were homogeneous in terms of age and gender (P = 0.387 and P = 0.785, respectively). A clinical history was compiled, and the following personal data were collected: age, gender, antecedents of cancer, smoking and alcohol consumption, exposure to chemical carcinogens or radiation, and diet.

Cytogenetic biomonitoring in oral leukoplakia

Report

conventional toothbrush, applying up to 20 circular movements. In the control group, the samples were collected from the cheek mucosa. Yellow-colored containers with a capacity of 30 ml were used, preparing 10 ml of buffer solution (0.1 0.01

M

Tris-HCl, 0.02

M

M

EDTA,

NaCl, pH 7; Sigma-Aldrich D9542,

St.Louis, MO, USA). The brushes were placed in the buffer solution and rotated several times to release the collected cells in the medium. The cell-containing solution was then transferred to centrifuge tubes and centrifuged for 10 minutes at 2000 rpm (581 g). After centrifugation, the supernatant was aspirated, and the cells were resuspended in 5 ml of a second buffer solution, followed by repetition of the centrifugation process.

Staining with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) A pipette was used to transfer 120–150 ll of cell suspension onto two slides. After drying, the latter were placed in an oven at 55 ºC for 15 minutes, followed by fixation with 50% methanol (Panreac SAU, E-08211, Barcelona, Spain) at 0 ºC for 15 minutes, and staining with DAPI (Sigma-Aldrich, D9542, USA) at a concentration of 200 lg/ml. DAPI is a fluorescent stain that strongly and specifically binds to DNA, thereby reducing the risk of false-positive readings (e.g. keratin bodies that could be wrongly taken to represent micronuclei (MN) if a non-DNA-specific stain such as the Giemsa stain were used).13 The slides were washed with Milli-Q water and examined with a Leika DRMB fluorescent microscope equipped with a DAPI band filter (excitation wavelength filter set [BP 340–380], dichroic filter RKP 400, and emission filters LP 425), under 9 100 magnification.

The study was carried out in the Department of Oral Medicine (Morales Meseguer Hospital, University of Murcia, Spain). All cases had been diagnosed between 2009 and 2011. The Ethics Committee of the University of Murcia approved the study protocol. All patients were fully informed of the purpose of the study, and informed consent was obtained in all cases.

Inclusion criteria Patients over 18 years old with a clinicopathological diagnosis of OL with mild dysplasia were included. The diagnosis of mild dysplasia was established by the same pathologist. The control group in turn consisted of subjects over 18 years old without diseases of the oral mucosa.

Identification criteria The criteria used for identifying the different types of cells and/ or nuclear anomalies were based on those established by Tolbert et al.14 These criteria classify the cells into categories, distinguishing between normal cells and abnormal cells based on cytological and nuclear features that are used to detect DNA damage, cytokinetic defects, and/or cell death. A more detailed description of the criteria used for identification of the types of cellular and nuclear alterations is provided below.11

Cells with MN These cells are characterized by the presence of a main

Exclusion criteria Exclusion criteria were patients declining to participate in the study and subjects under 18 years old.

nucleus and one or more smaller structures called MN. The MN are rounded or oval in shape, with a diameter between 1/3 and 1/16 that of the main nucleus. MN exhibit the same staining intensity and texture as the main nucleus. Most cells with MN

Sample collection and processing Oral exfoliated cells (ECs) were obtained from each subject by the same investigator. Before sample collection, the patients were instructed to rinse the mouth with water to eliminate the saliva and food particles. The lesions were then rubbed with a ª 2014 The International Society of Dermatology

have only one micronucleus, though some may contain two or more. Under normal conditions, MN have the same morphology as nuclei. The micronucleus should be located within the cell cytoplasm. MN are observed in differentiated cells, though they can also be identified in the basal cell layer (Fig. 1). International Journal of Dermatology 2014, 53, 1454–1459

1455

1456

Report

 nchez-Siles et al. Sa

Cytogenetic biomonitoring in oral leukoplakia

Figure 1 Differentiated cell with micronucleus

Cells with nuclear buds These cells show marked nuclear constriction, suggestive of the elimination of nuclear material through a budding process. The nuclear bud and nucleus are generally very close together and appear to be joined. The bud has the same morphology and staining characteristics as the nucleus, though its diameter can range from one-half to one-quarter that of the main nucleus (Fig. 2).

Binucleated cells These are cells with two main nuclei instead of a single nucleus. The nuclei are generally very close together and can even come into contact with each other. The morphological characteristics are generally similar to those seen in normal cells (Fig. 3).

Figure 3 Binucleated cell

Cells with condensed chromatin These cells present a striated nucleus in which the condensed chromatin aggregates stain more intensely. The chromatin in these cells is seen to accumulate in certain regions of the nucleus and become depleted in others.

Cells with karyorrhexis The nuclear chromatin of these cells aggregates more extensively than in the case of cells with condensed chromatin. An intensely staining mottled nuclear pattern is observed, indicative of nuclear fragmentation, leading to eventual disintegration of the nucleus.

Cells with pyknosis Pyknotic cells are characterized by the presence of a small nucleus, with dense nuclear material. The latter appears uniform but stains intensely. The nuclear diameter is usually about two-thirds that of a normal nucleus.

Cells with karyolysis In these cells, the nucleus has been completely devoid of DNA, producing a shadow or ghost-like image.

Figure 2 Cell with a nuclear bud (arrow) International Journal of Dermatology 2014, 53, 1454–1459

Cell quantification We initially examined 1000 cells per patient in order to identify the different types of cells: binucleated cells and cell death (cells with condensed chromatin, cells with karyorrhexis, and cells showing pyknosis and karyolysis). MN and nuclear buds were scored for 2000 cells or more. Both the differentiated cells and the basal layer cells were scored for MN. The two results were then combined to yield a global result.11 ª 2014 The International Society of Dermatology

 nchez-Siles et al. Sa

Cytogenetic biomonitoring in oral leukoplakia

Statistical analysis Analysis of the data was carried out using the SPSS version 12.0 statistical package (SPSS, Chicago, IL, USA). A descriptive study was made of each of the variables. The associations between qualitative variables were evaluated with the Pearson chi-squared test. The Student’s t-test for two independent samples was used to compare quantitative variables, with determination in each case of variance homogeneity. Statistical significance was accepted for P  0.05.

Results All 30 patients presented single OL lesions, distributed as follows: gums, 12 (40%); tongue, 9 (30%); cheek mucosa, 6 (20%); palate, 2 (6.6%); and floor of the mouth, 1 (3.3%). The mean age in the OL group was 59.10  9.27 years, vs. 61.97  15.46 years in the control group (P = 0.387). The gender distribution in the OL group was 11 males (36.67%) and 19 females (63.33%), while the control group comprised nine males (30%) and 21 females (70%; P = 0.785). In the OL group, 13 patients were smokers: nine smoked over 20 cigarettes a day; and four smoked between 10 and 20 cigarettes a day. Nine controls were smokers (three smoked more than 20 cigarettes a day, and six smoked 10–20 cigarettes a day). The proportion of alcohol consumers in the OL group was 20/30. Of these, 10 consumed alcohol daily (all being smokers), while 10 consumed alcohol on a sporadic basis. In the control group, six patients consumed alcohol occasionally (none on a daily basis). The frequency of MN in the patients with OL was significantly greater than in the control group (11.00  12.54 and 3.37  2.20, respectively; P = 0.002). The frequency of cells with nuclear buds was also significantly

Report

greater in the OL group (3.70  2.37 and 1.17  1.55, respectively; P = 0.019), in the same way as the incidence of binucleated cells (11.70  7.77 and 6.53  6.79, respectively; P = 0.008). In relation to cell death, the frequency of condensed chromatin (27.27  13.89) was significantly lower in the patients with OL than in the control group (51.30  23; P < 0.001). Cells exhibiting karyorrhexis were also significantly less frequent in the OL group (10.30  3.67) than among the controls (14.93  6.27; P = 0.001). In contrast, the incidence of pyknotic cells was greater in the control group than in the OL group, though in this case the difference failed to reach statistical significance (P = 0.026). The frequency of karyolytic cells was similar in both groups (Table 1).

Discussion The present study was designed to evaluate biomarkers of DNA damage (MN and/or nuclear buds), cytokinetic defects (binucleated cells), and cell death (condensed chromatin, karyorrhexis, pyknotic cells, and karyolysis) in ECs from patients with OL with mild dysplasia and a control group. These biomarkers have been associated with high cancer risk, neurovegetative disorders, and aging.11 In our study, we recorded a higher frequency of cells with MN in patients with OL with mild dysplasia than in the control group. Other authors have also reported a greater frequency of MN in patients with OL.8 The presence of MN is used as an indicator of chromosomal damage during the interphase and is associated with the first steps in carcinogenesis.15 Some investigators have reported an increase in the frequency of MN in the cells of patients with precancerous lesions when compared with control patients and in

Table 1 Comparison of DNA damage-cytokinetic defects, proliferative potential, and cell death between the OL group and control group (Students t-test) Cytogenetic variables

OL group (n = 30) mean  SD

DNA damage-cytokinetic defects MN 11.00  12.54 Nuclear buds 3.70  2.37 Binucleated cells 11.70  7.77 Proliferative potential Basal cells 10.27  4.62 Cell death Condensed chromatin 27.27  13.89 Karyorrhexis cells 10.30  3.67 Pyknotic cells 3.83  2.30 Karyolytic cells 55.20  15.11

Control group (n = 30) mean  SD

Odds ratio (95% CI)

P-value

3.37  2.20 1.17  1.55 6.53  6.79

2.32 (2.97–12.28) 1.04 (0.43–4.63) 1.88 (1.39–8.94)

0.002 0.019 0.008

7.27  4.15

1.13 (0.72–5.27)

0.011

51.30 14.93 2.43 55.30

   

23.00 6.27 2.44 18.80

4.94 1.32 0.61 4.41

( 33.92–14.14) ( 7.29–1.97) (0.17–2.62) ( 8.91–8.71)

< 0.001 0.001 0.026 0.982

SD, standard deviation; OL, oral leukoplakia; CI, confidence interval; MN, micronuclei ª 2014 The International Society of Dermatology

International Journal of Dermatology 2014, 53, 1454–1459

1457

1458

Report

 nchez-Siles et al. Sa

Cytogenetic biomonitoring in oral leukoplakia

cancer lesions compared with patients with precancerous lesions – thus suggesting that the presence of cells with MN can be taken as a biomarker of neoplastic progression.16 The frequency of nuclear buds and binucleated cells was significantly higher in the OL group than among the controls. The mechanism underlying bud formation is not clear, though the phenomenon may be related to the elimination of DNA or to DNA repair complexes.17 The significance of binucleated cells remains uncertain, though the phenomenon appears to indicate failed cytokinetics following the last step in nuclear division. The binucleated/mononucleated cell ratio could be an important biomarker indicating failed cytokinetics due to an increase in the aneuploid DNA rates.11 Precancerous lesions usually exhibit a greater presence of abnormal DNA (aneuploid DNA) compared with the typical diploidy of the cells of the normal epithelium.18,19 DNA aneuploidy is regarded as an indicator of malignancy.20 A lesser presence of condensed chromatin and of cells exhibiting karyorrhexis was recorded in the OL group – the differences with respect to the controls being statistically significant. Cell apoptosis is implicated in the etiology of a broad range of diseases, including cancer, where it plays an important role in carcinogenesis.21 Determination of the apoptotic index may have clinical applications by orienting the prognosis of potentially malignant lesions.22 It has been reported that the apoptotic index can increase, decrease, or remain unchanged in patients with OL compared with healthy subjects.23,24 In our study, all the studied types of cell death were less frequent than in the control group. In contrast, the presence of cells with karyolysis was similar in both groups. Apoptosis is regulated by a series of genes. The Bcl-2 gene family plays an important role in the regulation of apoptosis. Bcl-2 suppression favors oral carcinogenesis. Lako Loro et al. reported a decrease in Bcl-2 expression in oral epithelial dysplasias.25 Those cells that do not suffer apoptosis following DNA damage and which increase their proliferation rate can accumulate sufficient oncogenic events at a molecular level to result in cancer development. In addition to the clinical control, histopathological diagnosis, and surgical elimination of suspect lesions, these patients require regular long-term monitoring based on a rapid, simple, inexpensive, and scantly invasive technique. The technique described in the present study allows clinicians to monitor patients with precancerous oral lesions at risk of malignant transformation. CB is a sensitive, cost-effective, and scantly invasive technique offering information on the condition of the cells of the oral mucosa in patients with precancerous lesions, referred International Journal of Dermatology 2014, 53, 1454–1459

particularly to DNA damage and cell death – these being the main cellular alterations observed during carcinogenesis. References 1 Max Parkin D, Bray F, Ferlay J, et al. Global Cancer Statistics. CA Cancer J Clin 2005; 55: 74–108. 2 Mehrotra R, Gupta A, Singh M, et al. Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions. Mol Cancer 2006; 5: 11. 3 Acha A, Ruesga MT, Rodrˇguez MJ, et al. Applications of the oral scraped (exfoliative) cytology in oral cancer and precancer. Med Oral Patol Oral Cir Bucal 2005; 10: 95–102. 4 Pindborg JJ, Reichart P, Smith CJ, et al. World Health Organization: Histological Typing of Cancer and Precancer of the Oral Mucosa. Berlin: Springer, 1997. 5 Mithani SK, Mydlarz WK, Grumbine FL, et al. Molecular genetics of premalignant oral lesions. Oral Dis 2007; 13: 126–133. 6 Silverman S Jr, Gorsky M, Lozada F. Oral leukoplakia and malignant transformation. A follow-up study of 257 patients. Cancer 1984; 53: 563–568. 7 Papadimitrakopoulou VA, Hong WK, Lee JS, et al. Lowdose isotretinoin versus beta-carotene to prevent oral carcinogenesis: long-term follow-up. J Natl Cancer Inst 1997; 89: 257–258. 8 Mahimkar MB, Samant TA, Kannan S, et al. Influence of genetic polymorphisms on frequency of micronucleated buccal. Oral Oncol 2010; 46: 761–766.  9 V azquez-Alvarez R, Fern andez-Gonz alez F, G andara-Vila P, et al. Correlation between clinical and pathologic diagnosis in oral leukoplakia in 54 patients. Med Oral Patol Oral Cir Bucal 2010; 15: 832–838. 10 Spivack SD, Hurteau GJ, Jain R, et al. Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells. Cancer Res 2004; 64: 6805–6813. 11 Thomas P, Holland N, Bolognesi C, et al. Buccal micronucleus cytome assay. Nat Protoc 2009; 4: 825–837. 12 Iarmarcovai G, Bonassi S, Botta A, et al. Genetic polymorphisms and micronucleus formation: a review of the literature. Mutat Res 2008; 658: 215–233. 13 Nersesyan A, Kundi M, Atefie K, et al. Effects of staining procedures on the results of micronucleus assays with exfoliated oral mucosa cells. Cancer Epidemiol Biomarkers Prev 2006; 15: 1835–1840. 14 Tolbert PE, Shy CM, Allen JW. Micronuclei and other nuclear anomalies in buccal smears: a field test in snuff users. Am J Epidemiol 1991; 134: 840–850. 15 Bonassi S, Znaor A, Ceppi M, et al. An increase micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. Carcinogenesis 2007; 28: 625–631. ª 2014 The International Society of Dermatology

 nchez-Siles et al. Sa

16 Casartelli G, Bonatti S, De Ferrari M, et al. Micronucleus frequency in exfoliated buccal cells in normal mucosa, precancerous lesions and squamous cell carcinoma. Anal Quant Cytol Histol 2000; 22: 486–492. 17 Shimizu N, Kamezaki F, Shigematsu S. Tracking of microinjected DNA in live cells reveals the intracellular behaviour and elimination of extrachromosomal genetic material. Nucleic Acids Res 2005; 33: 6296–6307. 18 Onguru O, Celasen B, Gunhan O. Comparison of DNA ploidy and nuclear morphometric parameters with the conventional prognosis factors in transitional cell carcinomas. Tohoku J Exp Med 2003; 199: 141–148. 19 Femiano F, Scully C. DNA cytometry of oral leukoplakia and lichen planus. Med Oral Patol Oral Cir Bucal 2005; 10: 9–14. 20 Fabarius A, Hehlman R, Duesberg PH. Instability of chromosome structure in cancer cells increases exponentially with degrees of aneuploidy. Cancer Genet Cytogenet 2003; 143: 59–72.

ª 2014 The International Society of Dermatology

Cytogenetic biomonitoring in oral leukoplakia

Report

21 Wylie AH, Bellamy CO, Buba VJ, et al. Apoptosis and carcinogenesis. Br J Cancer 1999; 80: 34–37. 22 Cheng B, Rhodus NL, Williams B, et al. Detection of apoptotic cells in whole saliva of patients with oral premalignant and malignant lesions: a preliminary study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004; 97: 465–470. 23 Tanda N, Mori S, Saito K, et al. Expression of apoptotic signaling proteins in leukoplakia and oral lichen planus: quantitative and topographical study. J Oral Pathol Med 2000; 29: 385–393. 24 Ravi D, Ramadas K, Mathew BS, et al. De novo programmed cell death in oral cancer. Histopathology 1999; 34: 241–249. 25 Lako Loro L, Chrisitine Johannessen A, Karsten Vintermyr O. Decreased expression of bcl-2 in moderate and severe oral epithelia dysplasias. Oral Oncol 2002; 38: 691–698.

International Journal of Dermatology 2014, 53, 1454–1459

1459

This document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.

Cytogenetic biomonitoring in oral leukoplakia patients with mild dysplasia.

A study is made of DNA damage and apoptosis in a group of patients with oral leukoplakia (OL) with mild dysplasia...
232KB Sizes 1 Downloads 4 Views