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Int J Oral-Med Sci 6(2):81-90,2007 Original Article

The Cytological Findings of Oral Inflammatory , Planus and Leukopla- kia Coexisted with and without Candida : With Special Reference to Clinical, Histopathological,Immunohistochemical and Flow Cytometrical Analyses

Keisuke Nakazawa,Sisilia Fusi Fifita,and Kayo Kuyama

Department of Oral Pathology,Nihon University School of Dentistry at Matsudo,Matsudo,Chiba 271-8587,Japan

Correspondence to: Sisilia Fusi Fifita To evaluate the coexistence of Candida in potentially malignant oral

E-mail: fsisilia＠hotmail.com mucosal lesions: inflammatory lesions without features of leukopla- kia and (IL),lichen planus and using ex- foliative cytology(EC).This was evaluated and compared with clinical

findings,histopathology,immunohistochemistry and flow cytometrical

analysis(FCMA).In order to assess the coexistence of Candida with

,lichen planus and leukoplakia,the smears were taken

for Papanicolaou staining. To improve accuracy,PAS staining was

performed. Forty-nine cases of smears with biopsy specimens were

used for comparative study of EC and histopathology.Also, the im- munohistochemical activity of these cases was measured using p53,Ki -67 and COX-2.As for FCMA,the D.I.and P.I.were calculated for all

EC smears.One hundred and seventy-five cases of EC smears were

obtained from 40 normal tissue samples(control),inflammatory lesions (IL) 42, lichen planus 49and leukoplakia 44. Clinical findings

revealed the presence of whitish patches with in lichen

planus and leukoplakia,except for IL where erythema was present in

all cases.Overall,70 cases(40.0%)were confirmed by PAS to contain

Candida in the smear: IL 20, lichen planus 24, and leukoplakia 26with the coexistence rate of 47.6%,49.0% and 59.1%,respectively.

The cytological atypia in these cases was stronger than those without

Candida, but was different from dysplastic change.Forty-nine cases (28.0%)were available for immunohistochemical investigation where

both smear and histopathology specimens were taken.The mutation of

p53, cell proliferation Ki-67 and inflammatory index of COX-2; related with prostaglandlins, were also elevated in cases coexisted

with Candida. FCMA showed higher aneuploidy (D.I.)in cases with

Candida : 1.8 for IL, 2.1 for lichen planus and 2.2 for leukoplakia, respectively.Those without Candida were 1.5,1.9 and 2.0,respectively. The P.I.also showed the same tendency.Generally,Candida coexisted

with these lesions found in Papanicolaou and PAS stainings showed an

increased cytological atypia. Immunohistochemical results showed

higher positive reaction of p53,Ki-67 and COX-2 in Candida infected

diseases than those without Candida. The results indicated a strong

relationship with the FCM findings. The study concluded the DNA

Keywords: alterations in cases coexisted with Candida was likely to be considered inflammatory lesions, lichen planus, as irritative factor or promoter of dysplastic changes. The higher leukoplakia, Candida, exfoliative aneuploidy and proliferative index, and the immunohistochemical cytology,clinico-histopathology,im- results gave significant indications that candidal infection may disturb munohistochemistry,flow cytometry the DNA cellular content. Int J Oral-Med Sci 6(2):81-90,2007

coexisted with Candida and cases without.To over- The relationship between Candida infection and come the‘third’aforementioned problem of,by using the risk for malignant transformation has been an the FCMA couple to EC, PAS, and immunohisto- interest in the study of oral candidosis(1). Candida, chemistry.Flow cytometry(FCM)is the scanning of the promoter or initiator of malignant transforma- a cytology sample and nuclear staining,which facili- tion has also been discussed,but the exact histopath- tates the identification of special groups of tumor ological phenomenon is still unknown in inflamma- cells or an abnormal number of chromosomes; both tory lesions without features of leukoplakia and are associated with reactive or inflammatory lesions, lichen planus(IL),lichen planus and leukoplakia(2). epithelial dysplasia and . It has been

Whether the microorganisms are causally involved reported that DNA content analysis provides prog- in the development of leukoplakia or just secondary nostic information eliminating the subjective compo- invaders are a matter still being debated (3-5). nent involved in routine microscopic decision-mak- Candida has also been identified in lichen planus and ing and EC (20-22). its involvement in malignant transformation in some Therefore in this study, an assessment of FCM lichen planus is still obscure(6,7).Hence,it is shown approaches to determine the DNA aneuploidy of IL, that this microorganism may cause aggravation of lichen planus and leukoplakia coexisted with Can- existing leukoplakia and lichen planus (8-10). dida and those without,and compare to EC and the

Krog et al.,have demonstrated higher nitrosation immunohistochemical findings. potentials of candidal organisms isolated from leuko- plakia,may thus play a role in carcinogenesis proc- ess (11).This may occur by way of interaction with Smear specimens other carcinogens or directly on oral epithelial cells. EC cases of IL,lichen planus and leukoplakia were

Several species of Candida,especially C.albicans can selected during oral examinations at the time of their cause a variety of oral pathologies,of which thrush first visit to Nihon University School of Dentistry at is the most commonly encountered (12).It exists as Matsudo.From each case,cells were exfoliated using an oral commensal in 40-60% of humans but become cytobrush (Medscand Medical,CoperSurgical Com- opportunistic pathogens when there is immunosup- pany,Berlin,Germany).The protocol was approved pression,either local or systemics(13).Local changes by the Committee on Studies Involving Human Being favour their proliferation and adherence to oral of Nihon University School of Dentistry at Matsudo mucosa,and such changes include poor , (E-5.002).Informed consent was obtained from all presence of surface roughening and whitish patchies patients before taking the cytological smears. (14).The atypical changes occurring in epithelial cells and Candida could be diagnosed in the smears Clinical assessment using Papanicolaou and PAS stains, but histopath- The clinical assessments of IL, lichen planus and ological examination is difficult due to laboratory leukoplakia were according to WHO clinical classifi- handling of the biopsy specimen(15-19). cation in 1992 and 2005 (23, 24).Leukoplakia is a

Our study was considered: First, to evaluate the white of the that cannot be char- frequency and distribution of Candida in smears of acterized as any other definable lesion. Lichen inflammatory lesions without features of leukopla- planus is a chronic mucocutaneous immune inflam- kia and lichen planus were recorded as IL, lichen matory condition and consists of papular,reticular, planus and leukoplakia. Second, to correlate the plaque like, atrophic, erosive and bullous types. presence of Candida in smears and biopsies in cases Other mucosal inflammatory lesions with no features where both were provided. Third, to analyse flow of leukoplakia and lichen planus were recorded as cytometrically the IL,lichen planus and leukoplakia IL. Int J Oral-Med Sci 6(2):81-90,2007

Cytological assessment of smears tochemical examinations (Figs.1a,2a,3a).

Each patient’s mouth was first rinsed with water once to remove food debris and moisten the surface. Immunohistochemical assessment

To obtain a smear,the cytobrush was rolled at least Sections of all 49 cases were deparaffinized in

5 times with gentle pressure on the mucosal surfaces. xylene and dehydrated in Tris-buffered saline(TBS, The clinician took two sets of smears for each Takara, Japan, Code No.T903 in 0.05 M sodium patient.The first sets were smeared on labeled glass citrate,pH-7.6).For antigen detection,the envision＋ slides,immediately fixed in 95% ethyl alcohol,and labeled streptavidin-biotin (LSAB 2 kit,Dako A/S, subsequently stained by the Papanicolaou method Canada) technique was used. Primary anitibodies, (25).One slide glass was reserved for PAS staining. directed against the following antigens,were used at

The diagnoses were made according to the dilutions indicated: p53 (DO-7, 1: 50; Dako

Papanicolaou’s classification and General Rules for Cytomation, Canada), Ki-67 (MIB-1, 1: 50; Dako

Clinical and Pathological Study of Uterine Cervical A/S, Denmark) and COX-2 (CX-294, 1: 50; Dako

Cancer in Japan (26).Diagnoses were only classified Cytomation,Canada).To improve detection,depar- as: Class I: no neoplastic cells identified; Class II: affinized sections were pretreated by microwave atypical cells present but no neoplastic cells heating. Peroxidase activity was visualized using identified.To improve accuracy,PAS staining was diaminobenzidine.Finally,all sections were counter- added for detecting Candida. Cytological diagnoses stained with Mayer’s hematoxylin.Positive controls were done by one cytologist and two pathologists. consisted of specimens of

The coexistence rate was investigated as the percent- for Ki-67, p53 and COX-2. For evaluation of the age of cases where Candida was present in both immunostaining technique, as a negative control,

Papanicolaou and PAS stainings.PAS staining was mouse IgG1 (COX-2, Ki-67)and IgG2b (p53) were performed to confirm the presence or absence of used instead of the primary antibodies during the

Candida. staining procedure.All were examined in at least 700

The second set of smears was performed in the cells in 8 random microscopical fields and the posi- identical site and was immediately placed into a 1.5 tive stained nuclei were counted using a high power ml conical tube containing di-methyl sulfoxide in (×40)objective lens. sucrose-citrate buffer for FCMA (CycleTESTTM

PLUS DNA Reagent KIT,Becton Dickinson,Califor- Flow cytometrical assessment nia, USA). Forty volunteers with normal buccal All smear samples were analyzed using FCM. mucosal smears were used as the control with the Scrapings of the epithelial cells were obtained direct- mean age of 40.8±10.4 years, and composed of 25 ly from the lesions by means of a cytobrush. To females and 15 males.These were non-smokers,non prepare single-cell suspensions for FCM, the epith- -denture wearers,had not consumed any spicy or hot elial cells were stained with propidium iodide,as it foods or rinsed with mouthwash before smears were has been successfully employed previously on FCMA taken. The same procedure was performed for the of head and neck tumors(27,28).Cell cycle analyses control smears using Papanicolaou method, PAS were performed using a Cycle TEST PLUS DNA staining and FCMA. Reagent Kit (Becton Dickinson and Company, California,USA)and data was analyzed using Cell- Histopathological assessment Quest software and ModFit LT (Verity Software

Forty-nine cases where smears with tissue speci- House,California,USA).The cellular DNA content mens either from biopsy or operational materials of 5,000 to 15,000 cells was measured,recorded,and were stained with hematoxylin and eosin,and PAS displayed in a single parameter DNA-content histo- and subjected to histopathological and immmunohis- gram.In these series of studies,the DNA content was Int J Oral-Med Sci 6(2):81-90,2007 evaluated for ploidy,in relation to cell cycle and a Cytological and PAS findings summary of DNA index(D.I.)(mean±standard devi- The cytological diagnoses are also summarized in ation),and proliferative index (P.I.)(%)was record- Table 1.No cellular or nuclear abnormalities were ed. The FCM results were compared to all observed in the smears obtained from the control cytological and immunohistochemical findings of group. Based on our EC results, 22.9% were from cases coexisted with Candida and ones without. normal mucosa (control)and 77.1% were diagnosed

ClassⅡ : IL (24.0%),lichen planus (28.0%),and leu- Statistical method koplakia (25.1%).When the Papanicolaou and PAS

Immunohistochemical findings and D.I. and P.I. findings were compared, the coexistence rates of were analyzed statistically using Welch’s test (p＜ Candida in IL, lichen planus and leukoplakia were

0.05)for cases having Candida and the ones without. 47.6%,49.0% and 59.1%,respectively(Figs.1b,2b,3b

and Figs.1c,2c,3c). Overall,nuclear changes in these IL,lichen planus

Patient population and clinical findings and leukoplakia were limited to slight enlargement,

The study population consisted of 175 patients who in some instances with mild hyperchromatism and had taken cytological smears with clinical diagnoses slightly increased N/C ratio. Regarding IL, the of no disease; normal(control),IL,lichen planus and inflammation atypia was stronger in leukoplakia.The mean age of the patients was 50.3± than the cases without Candida ; however, the fea- 12.3 years (range 28-85) and consisted of 51.0% tures were different from dysplastic change. females and 49.0%males.The patient population and major clinical findings of 175 samples are summar- Histopathological findings ized in Table 1. Overall,49 cases were available for comparative

Generally, IL consisted of decubitus , gin- study of cytology and histopathology where Candida givitis, periodontal having erythematous was found in 32 cases(65.3%).The histopathological areas and whitish patches that were easily removed findings matched the cytological results with no on smearing.Lichen planus and leukoplakia showing dysplastic epithelium. Among 15 IL cases with erythematous areas between white patches without hematoxylin and eosin and PAS evidence of Candida, dysplasia were whitish reticular,with striae appear- 10 had histopathological findings,while lichen planus ing for the former and white patches for the latter, and leukoplakia had 12 out of 18 and 10 out of 16 and showed mild to moderate irregular keratinized cases, respectively.In addition, 17 cases were his- mucosae. There were no severe non-homogenous topathologically matched the Papanicolaou and PAS whitish lesions included in this study. results which showed no evidence of Candida ; 5

Table 1. Patient population according to clinical diagnoses,Papanicolaou and PAS stainings

Cases Main clinical features Presence of Candida Presence of Candida

Pap & PAS H.E& PAS No./Total

No./Total(%) of H/PATH(%) Normal(Control) firm and pale pink buccal mucosa 0/40 0/0

IL decubitus ,,periodontal ,erythema 20/42(47.6) 10/15(66.7) Lichen planus whitish reticular patches with striae,erythema 24/49 (49.0) 12/18(66.7) Leukoplakia flat and thin to thicker whitish patches,erythema 26/44(59.1) 10/16(62.5) Total 70/175(40.0) 32/49 (65.3) : total of each lesion (IL: inflammatory lesions,lichen planus and leukoplakia) : some were scraped off easily on smearing : total number of histopathological cases Int J Oral-Med Sci 6(2):81-90,2007

Fig.1a. High-power microscopic image of an inflammatory lesion showing Candida hyphae.Candida is seen on the surface of epithelial cells and within the epithelial cells(arrowheads,hematoxilin-eosin,original magnification ×400). Fig.1b. PAP staining shows Candida hyphae.The smear contains squamous cell with slight enlargement of the nuclei,fine chromatin pattern and the atypia is stronger but different from dysplasia(arrowhead,Pap’s ×400). Fig.1c. PAS staining shows the yeast cells,budding spores and hyphae(PAS ×400).

Fig.2a. Microscopic image of lichen planus (hematoxilin-eosin,original magnification ×200). Fig.2b. PAP staining shows Candida hyphae intermingled between the squamous cells.The smear contains squamous cell with light enlargement of the nuclei,mild hyperchromatism and slightly increased N/C ratio(arrowhead,Pap’s ×400). Fig.2c. PAS staining shows the yeast cells,budding spores and hyphae(PAS ×400).

Fig.3a. Microscopic image of leukoplakia(hematoxilin-eosin,original magnification ×200). Fig.3b. PAP staining shows Candida hyphae.The smear contains squamous cell with light enlargement of the nuclei,mild hyperchromatism,slightly increased N/C ratio,strong orange cytoplasms and stronger atypia(arrowhead,Pap’s ×400). Fig.3c. PAS staining shows the yeast cells,budding spores and hyphae(PAS stain ×400). cases in IL and 6 cases each from both lichen planus without Candida involvement.All histopathological and leukoplakia. specimens were immunostained with p53,Ki-67 and

COX-2.Table 2 showed the comparison among all

Immunohistochemical findings the cases where Candida coexisted and ones without

Immunohistochemical markers were used to evalu- (Figs.4a-4c,5a-5c,6a-6c),(Figs.4d-4f,5d-5f,6d-6f). ate in tissue sections from cases with Candida and There was an enhanced level of proliferation as Int J Oral-Med Sci 6(2):81-90,2007

Table 2. Immunohistochemical findings of cases with and without Candida

IL (%) Lichen planus (%) Leukoplakia(%) No.of H/PATH Cases 15 18 16

Candida ＋ － ＋ － ＋ －

p53 15.2 9.2 22.7 10.5 30.6 15.3

Ki-67 17.8 12.4 28.6 24.5 21.5 17.2

COX-2 20.3 11.8 30.3 22.7 8.5 3.4

IL: inflammatory lesions : histopathological cases where 5 out of 15,6 out of 18 and 6 out of 16 showed no Candida in the H.E/PAS stains ＋ : % of positive cells in cases with Candida － : % of positive cells in cases without Candida

Figs.4a-4c. Some squamous cells of inflammation stain positive for Ki-67,p53 and COX-2.All cases have Candida in smears showing in PAP and PAS stains (original magnification ×400). Figs.4d-4f. A few cells are staining positive.These cases reveal no Candida in PAP and PAS stains (original magnification ×200).

Figs.5a-5c. Some squamous cells of lichen planus stain positive for Ki-67,p53 and COX-2.All cases have Candida in smears showing in PAP and PAS stains (original magnification ×400). Figs.5d-5f. A few cells are staining positive.These cases have no Candida in the smears showing in PAP and PAS stains (original magnification ×200).

Figs.6a-6c. Some squamous cells of leukoplakia stain positive for Ki-67,p53 and COX-2.All cases have Candida in smears showing in PAP and PAS stains (original magnification ×400). Figs.6d-6f. A few cells are staining positive.These cases have no Candida in the smears showing in PAP and PAS stains (original magnification ×200). Int J Oral-Med Sci 6(2):81-90,2007 measured by p53 mutation and Ki-67.Overall, the the highest mean D.I. and P.I. values. There were results showed that all cases with Candida had statistically differences between the D.I.and P.I.of higher proliferative index and positively stained cells IL, lichen planus and leukoplakia without Candida compared to ones without.Further,COX-2 immuno- compared to normal (control)ones. histochemical expression in these cases was also high.The differences were significant (p＜0.05). Regarding epidemiological studies, as early as

Flow cytometrical findings 1960’s,reported the changes in leukoplakia related to

All 175 cases were FCM analyzed. Cases were Candida infection by producing increased hyperker- divided into two groups and formed the basis of this atosis and mitotic activity(10,11).This corresponds analysis.Group 1 consisted of cases where Candida well with the present findings,where an increase in coexisted in both Papanicolaou and PAS staining ; proliferative activity found immunohistochemically

IL(n＝20),lichen planus(n＝24)and leukoplakia(n＝ compared to cases without Candida.The recognition

26).Group 2 consisted of cases where Papanicolaou of these fungal pathogens capable of initiating leuko- and PAS staining showed no Candida ; IL (n＝22), plakia was taken into consideration.However,there lichen planus (n＝25)and leukoplakia (n＝18).The is a longstanding discussion whether Candida infec-

D.I.mean value of control cases was 1.0±0.3,and the tion is a cause of leukoplakia or if it is a super- mean P.I.value was 52.6%. imposed infection in a pre-existing lesion (3,12).On

the other hand,Zeng et al. had studied the adhesion

Group 1 with Candida of Candida albicans to epithelial cells resulted and

Table 3 summarizes the results of D.I. and P.I. attributed to the occurrence and progression of ero- mean values.Regarding the IL coexisted with Can- sive lichen planus (29). dida,the mean D.I.and P.I.values were higher than Bouquot mentioned that a higher malignant trans- the control ones. formation rate reported in Candida-infected leuko- As for the IL,lichen planus and leukoplakia with plakias: 3 times as many Candida leukoplakia cases

Candida the mean D.I. and P.I. values were in- developed carcinoma than leukoplakia without Can- creased; with leukoplakia associated with Candida dida ; 30% and 10%, respectively (30). Moreover, being the highest. There were statistically differ- from the 1990’s, carcinogenesis experiments in ani- ences between the D.I.and P.I.of lichen planus and mal studies indicated that C.albicans had produced leukoplakia with Candida and the control ones(p＜ oral mucosal carcinomas in rats(4,11,31).The yeast

0.05).The mean P.I.values also showed an increase is capable of producing keratotic plaque,and Can- in tendency with the coexistence of Candida. dida has been shown to produce carcinogen

nitrosamines,a high nitrosation endogenous carcino- Group 2 without Candida gens.Therefore,the study was specific for IL,lichen

Among all cases without Candida,leukoplakia had planus and leukoplakia because each of these has its

Table 3. Values for D.I.and P.I.of cases with and without Candida

Normal(Control) D.I.＝1.0±0.3(0.1－1.2) P.I.＝52.6% Group 1.Cases with Candida Group 2.Cases without Candida

D.I.(Range) P.I.(%) D.I.(Range) P.I.(%) IL with Candida 1.8±0.5(1.0－2.9) 67.5 IL 1.5±0.4(1.0－2.5) 60.4

Lichen planus with Candida 2.1±0.5(1.7－3.3) 71.9 Lichen planus 1.9±0.5(1.0－2.9) 64.9

Leukoplakia with Candida 2.2±0.5(1.8－3.5) 75.3 Leukoplakia 2.0±0.6(0.9－3.6) 65.4

IL: inflammatory lesions Int J Oral-Med Sci 6(2):81-90,2007 own level of risk.Further,the use of EC in IL,lichen findings should lead to the follow-up of these planus and leukoplakia that could be scraped to patients.Overall,nuclear changes in these IL,lichen check for the presence of Candida was also amenable planus and leukoplakia were not dysplastic. The

(5).In some cases with non-homogenous leukoplakia, presence or invasion of Candida in these lesions as a there is superficial invasion by fungal hyphae and it secondary event or the causal remains uncertain,but has been demonstrated that these have a higher risk the authors are certain of Candida inducing changes of malignant transformation(9,19).These non-inva- exert in epithelial cells was established. sive hyphae and fungi could be diagnosed in the Our FCM results of Group 1 that coexisted with smears and in the culture,but could be lost during the Candida showed an increased D.I. and P.I. values laboratory handling of the biopsy specimen.There- from those without Candida. FCMA was useful in fore,EC is convenient for research and our study use determining the cell biological changes in these oral

FCM combined with histopathology. mucosal lesions that cannot be detected by EC and

Our results are based on the 175 cases used for this PAS methods.The increasing DNA aneuploidy and study and confirm previous evidence of the coexis- P.I.proportion to presence of Candida in these cases tence of Candida in IL,lichen planus and leukoplakia was highlighted in this FCMA.It could be due to the in the oral cavity(1).The authors supported the fact high turnover rate inherent in the oral epithelium or that the expression of these in the early stages of oral increased cell proliferation due to presence of Can- tumorigenesis was associated with the presence of dida as an irritation or promotion of nuclei changes.

Candida (1, 14).Further, this is the first study to This provides interesting results, facilitating more assess flow cytometrically and immunohisto- detail investigation of disease progression as obser- chemically the IL, lichen planus and leukoplakia ved in cases coexisted with Candida. without dysplasia that coexisted with Candida infec- Regarding Group 2 without Candida showed in- tion.It appears that the methods are sensitive to the creased mean values of D.I.and P.I.from those of the presence of Candida and may be there is a role for control ones.The observation that these cases were

Candida in the development of pathological condi- higher than in control ones may indicate the com- tions, cytological changes, increased D.I., and mencing of development of new disease.Also,in this proliferative indices.On the other hand,although the study there was a trend for increased D.I.,P.I.from present study provides supporting evidence for a role IL to lichen planus and leukoplakia although the of Candida in these lesions it may also be possible differences were small between leukoplakia and that the presence of Candida may reflect a changed lichen planus and not statistically significant.There- local environment that allows for the appearance of fore,the FCM results correlated and matched the EC this commensal microorganism. diagnoses of the IL, lichen planus and leukoplakia, The most common lesions in our material were and the results represented the aneuploidies in these lichen planus and leukoplakia.Leukoplakia had the lesions,as the EC classification increased from the most cases coexisted with Candida followed by control cases.FCMA from this study begins to estab- lichen planus and IL.Thus the finding of Candida in lish links between normal control and changes these IL proved the usefulness of EC in detecting identified in cases without Candida to cases coexist- these microorganisms,and showed its importance in ed with Candida. It further showed the detection of overcoming confusing diagnoses according to inflam- higher D.I. and P.I. in these lesions compared with matory reactive changes. Regarding lichen planus controls gave a significant result. and leukoplakia,no dysplastic cells were identified. Moreover, the results also related better to the

In addition, the IL with Candida, the inflammation immunohistochemical findings of cases coexisted atypia was stronger than ones without,however,the with Candida.Immunohistochemically,p53 mutation features were different from dysplastic change.The showed higher proliferative activity and demonstrat- Int J Oral-Med Sci 6(2):81-90,2007 ed more frequently in cases coexisted with Candida method for cases coexisted with Candida is accept- than the cases without.The higher values indicated able. that the presence of Candida might be affected the

DNA alterations is considered to be early epithelial changes of dysplasia. Hence, it demonstrated the The authors are grateful to Professor H. expression of p53 and high rates of cell proliferation Yamamoto (Department of Oral Pathology,Nihon as deregulation of the cell cycle progresses similar to University School of Dentistry at Matsudo)for his this study(32).As with Ki-67,the same tendency was direction and helpful advice,and appreciate Profes- seen.Those with Candida had increased proliferative sors H.Kozawa, T.Kondoh and M.Yamamoto activity than ones without,thereby proving the acti- (Departments of Histology,Cytology and Develop- vation of cell cycle due to the presence of these mental Anatomy,Maxillofacial Surgery,Microbiol- microorganisms. Our study suggested that the in- ogy and Immunology,respectively,Nihon University creased detection of p53 and Ki-67 in cases coexisted School of Dentistry at Matsudo) for reviewing the with Candida indicated DNA alterations and deserve manuscript.The authors also acknowledge the staff particular attention because it might offer further of our department for their excellent support. understanding of these lesions. Interestingly, there The study was supported by Suzuki Memorial was a strong correlation between p53 positivity and Grant of Nihon University of Dentistry at Matsudo

Ki-67 markers in these cases appear to be attributed and the Ministry of Education, Culture Sports,Sci- to acceleration of the cell cycle and lesion develop- ence and Technology,Japan,Grant-in-Aid for 2004- ment.Regarding, the COX-2 immunoreactivity,our 2006,16591839,Scientific Research(C). data showed that those coexisted with Candida in- creased P.I.,thus confirming the inflammatory phe- 1. McCullough M,Japer M,Barrett AW,Bain L,Speight nomenon and increase PGE2 synthesis may affect or PM, Porter SR : Oral yeast carriage correlated with accelerate the growth of Candida (33).In addition, presence of oral epithelial dysplasia.Oral Oncol,38: many studies have indicated that COX-2 expression 391-393,2002. in carcinogenesis and a few reports on dysplasia and 2. Silverman S : , 4th ed.,B.C Decker Inc. carcinoma (34).In the present study,the expression London; 1998.p.31-36. 3. Holmstrup P, Bessermann M : Clinical, therapeutic, of COX-2 was increased in the site of IL, lichen and pathogenic aspects of chronic oral multifocal planus and leukoplakia coexisted with Candida than candidiasis.Oral Surg Oral Med Oral Pathol,56: 388 ones without. -395,1983. Our EC findings detected the presence of Candida 4. Renstrup G : Occurrence of Candida in oral leukopla- - in some cases and further confirmed by PAS findings kias.Acta Pathol Microbiol Scand,78: 421 424,1970. 5. Banoczy J : Follow-up studies of oral leukoplakia.J and correlated with the histopathological findings. Max-Fac Surg, 5: 69-75,1977. The clinical findings revealed the presence of white 6. Krog P, Thorn JJ, Pindborg JJ: Yeasts species and patches, which were thought to harbor Candida biotypes associated with oral leukoplakia and lichen during smear taking. As a result of this,the smear planus. Oral Surg Oral Med Oral Pathol,63: 48-54, 1987. revealed the Candida couple to PAS staining but 7. Tyldesley WR : Malignant transformation in oral

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