Journal of Investigative and Clinical Dentistry (2011), 2, 176–183
ORIGINAL ARTICLE Oral Pathology
Actual proliferating index in oral squamous cell carcinoma and leukoplakia Abhay R. Chandak1, Amol Ramchandra Gadbail2, Minal S. Chaudhary2, Shweta A. Chandak3 & Ritesh Wadhwani4 1 Department of Oral and Maxillofacial Pathology and Microbiology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India 2 Department of Oral and Maxillofacial Pathology and Microbiology, Datta Meghe Institute of Medical Sciences, Sharad Pawar Dental College and Hospital, Sawangi (Meghe), Wardha, Maharashtra, India 3 Department of Pediatric and Preventive Dentistry, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India 4 Department of Oral and Maxillofacial Pathology and Microbiology, Jamanlal Goenka Dental College and Hospital, Akola, Maharashtra, India
Keywords actual proliferation index, argyrophilic nucleolar organizer region count, Ki-67 labeling index, oral premalignant lesion, oral squamous cell carcinoma. Correspondence Dr Amol Ramchandra Gadbail, Department of Oral and Maxillofacial Pathology and Microbiology, Datta Meghe Institute of Medical Sciences, Sharad Pawar Dental College and Hospital, Sawangi (Meghe), Wardha 442001, Maharashtra, India. Tel: +91-97661-35691 Fax: +91-071-5228-7713 Email: [email protected] Received 27 December 2010; accepted 29 January 2011. doi: 10.1111/j.2041-1626.2011.00057.x
Abstract Aim: To examine the possible association between epithelial proliferation and disease progression in the oral mucosa using the actual proliferation index. Methods: The actual proliferation index was measured by the Ki-67 labeling index and argyrophilic nucleolar organizer region count per nucleus. Immunohistochemistry was carried out for Ki-67 by using the molecular immunology borstel-1 clone in 20 leukoplakias, 20 oral squamous cell carcinomas, and 10 normal oral mucosae. Results: The argyrophilic nucleolar organizer region count per nucleus, Ki-67 labeling index, and actual proliferation index were significantly higher in oral squamous cell carcinoma, followed by leukoplakia and normal oral mucosa. Leukoplakia with dysplasia showed a significantly higher Ki-67 labeling index and actual proliferation index, compared to leukoplakia without dysphasia. There was a significant correlation of Bryne’s histological malignancy grading with the argyrophilic nucleolar organizer region count and the Ki-67 labeling index. There was a significant positive correlation between the argyrophilic nucleolar organizer region count and the Ki-67 labeling index among all groups. Conclusions: Leukoplakia or suspected epithelial dysplasia should be stained for argyrophilic nucleolar organizer regions and Ki-67. The actual proliferation index is not only useful as a prognostic factor, but could also be a promising treatment determining modality for patients with premalignant and malignant lesions.
Introduction Oral cancer holds eighth position in cancer incidence worldwide. A geographic variation in the incidence of oral squamous cell carcinoma (OSCC) exists. In India, OSCC ranks first among all cancers in males, and third in females. Incidence rates vary from seven to 17/100 000 people annually. Based on cancer registry data, it is estimated that 75 000–80 000 new cancer cases develop annually in India.1 176
Squamous cell carcinoma is the most common malignant neoplasm of the oral cavity, accounting for more than 90% of all oral malignancies. It is usually preceded by premalignant lesions. The identification of these premalignant lesions through clinical, morphological, and molecular means helps in the early detection and treatment of OSCC. Leukoplakia is the most common oral premalignant lesion.2 Oral premalignant lesions are known to represent an increased risk of oral cancer development, with quoted malignant transformation rates ª 2011 Blackwell Publishing Asia Pty Ltd
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varying from 6.6% to 36%.3 Changes in the proliferative capacity of oral premalignant lesions might indicate early neoplastic changes and the risk of malignant transformation.4 It is generally agreed that lesions exhibiting oral epithelial dysplasia are at increased risk of malignant transformation. Epithelial dysplasia is characterized by a number of cellular and tissue alterations that reveal an alteration of cell maturation in the epithelium. However, it has been noted that there might be subjective variation in the diagnosis and grade of the same sample of epithelial dysplasia by different pathologists. Therefore, markers of cellular proliferation might help to establish more objective diagnostic criteria. Previous studies on the proliferative characteristics of oral epithelia of precancer and cancer have been performed by using various methods.5,6 Among those most often used, immunohistochemical staining using the molecular immunology borstel-1 (MIB-1) antibody against the Ki-67 antigen has emerged as a marker of choice. In terms of cell biology, the proliferative state reflects the percentage of a proliferation-associated population at any particular time.7 The Ki-67 expression assesses the state of cell proliferation, that is, growth fraction (the proportion of cells committed to the cell cycle). The Ki-67 human nuclear antigen is expressed during the G1, S, G2, and M phases in the cell cycle, but is absent in the quiescent G0 phase.8 The proliferation rate represents measurements in which a cell-cycle phase transit time is calculated to allow the estimation of a doubling time.7 This can be evaluated by an argyrophilic nucleolar organizer region (AgNOR) parameter. Interphase AgNOR are structural and functional units of the nucleolus, in which all of the components necessary for ribosomal RNA synthesis are located. The number of interphase AgNOR are strictly related to rRNA transcriptional activity, and in continuously-proliferating cells, to the rapidity of cell proliferation.9 Therefore, the actual proliferative activity of a lesion can simply be expressed as: Actual proliferative activity = Ki-67 or MIB-1 scores · AgNOR quantity.6,10 In the present study, we aimed to examine the possible association between actual proliferative activity and disease progression in the oral mucosa using the Ki-67 labeling index (LI) and AgNOR count to determine true proliferative index. We further compare the clinical parameters and histological grading of OSCC in the context of cell proliferation. Methods The present study was carried out at the Department of Oral Pathology and Microbiology, Sharad Pawar Dental College, Wardha, India. The study included 20 histopathologically diagnosed OSCC patients, 20 oral leukoplakia ª 2011 Blackwell Publishing Asia Pty Ltd
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patients, and 10 normal oral mucosa patients. In the study, patients with oral leukoplakia had white and/or red lesions with no other etiological factor, except a habit of tobacco smoking, as well as tobacco quid and betel nut chewing. The clinical information was recorded for OSCC and leukoplakia following a definite format. Written consent was obtained from all patients. Approval for the study was given by the Institutional Ethical Committee of Datta Meghe Institute of Medical Sciences, Sawangi (Meghe), Wardha, India. The study was performed on tissues fixed in 10% neutral buffered formalin, paraffinembedded tissue. The sections were cut serially to a 4-lm thickness for AgNOR staining, and 5 lm for immunohistochemistry to evaluate the expression of the Ki-67 antigen. Leukoplakia samples were divided into two groups: with and without dysplasia. In OSCC, clinical TNM staging and histological malignancy grading (World Health Organization [WHO] and Bryne’s) was carried out. Bryne’s histological malignancy grading was performed on most anaplastic fields, and cases were separated into three categories, which had significantly different prognostic groups, as suggested by Bryne et al.11 Procedure AgNOR staining method Solution A Two grams (2% w/v) of gelatin powder was added to 100 mL distilled water, with the temperature maintained at 60–70C over a water bath. Later, 1 mL formic acid (1%) (H.COOH = 46.03; Qualigens Fine Chemicals, Mumbai, India) was added and the mixture was stirred thoroughly. Solution B Twenty-five grams of silver nitrate (AgNO3 = 169.87; Qualigens Fine Chemicals) was added to 50 mL distilled water. After the dissolution, the liquid was filtered with #1 Whatman filter paper (Qualigens Fine Chemicals). Working solution Twenty-five milliliters of solution A was mixed with 50 mL of solution B to obtain a resultant 75 mL of working solution. Staining was carried out according to the International Committee on AgNOR Quantitation.12 Slides were hydrated and heated in a 750-W microwave oven for 10 min in 10 mm sodium citrate buffer (pH 6.0). The sections were then washed three times with double-distilled water for 5 min each, and incubated in the dark for 20–25 min in a working solution at room temperature. The slides were washed in running doubledistilled water for 10–15 min. 177
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Quantification of AgNOR All the slides were examined by light microscopy at a 1000· magnification. In each section, a minimum of 200 nuclei were examined, and the mean number of AgNOR per cell was calculated. Counting was done according to the criteria of Giri et al.13 By careful focusing, clearlydefined dots of AgNOR within nucleoli and small AgNOR outside nucleoli were counted. AgNOR appears as dark brown or black intranuclear bodies against a yellowish background. When AgNOR were found lying in clusters and were not individually resolvable, the cluster of AgNOR was counted as a single AgNOR. Lymphocytes in the lamina propria were used as an internal control. For normal epithelia and leukoplakia, basal and adjacent parabasal layers were evaluated. In well-differentiated squamous cell carcinomas, keratinized cells were avoided because nuclear pyknosis obscure the AgNOR. Mean AgNOR per cell were calculated for each case, and group means were derived. Immunohistochemistry for detecting the Ki-67 antigen For immunohistochemistry, the peroxidase detection system (streptavidin–biotin detection system; Novocastra kit; Novocastra Laborotories, Newcastle-upon-Tyne, UK) was employed. The sections were deparaffinized with xylene and hydrated. The slides were heated in microwave oven for 10 min in 0.01 m sodium citrate buffer (pH 6.0) for antigen retrieval and bench cooled for 20 min; the cycle was then repeated. Endogenous peroxidase activity was blocked by incubating the section with 3% H2O2 in methanol for 30 min. The sections were then washed three times with gentle shaking in phosphate-buffered saline (PBS) for 5 min each. To prevent non-specific reactions, the sections were incubated with 10% serum for 10 min. Prediluted Ki-67 antibody (clone MIB-1; Dako, Glostrup, Denmark) was incubated at room temperature in a humidifying chamber for 60 min, and then at 4C overnight. The hyperplastic lymph node was used as a positive control. One section from the positive control was used as the negative control by omitting the primary antibody and by incubating with the serum. After the primary antibody and antigen reactions, the sections were rinsed in PBS three times for 10 min each. The secondary antibody (biotinylated rabbit antimouse immunoglobulin G) was incubated at room temperature in a humidifying chamber for 30 min. After washing the sections three times in PBS for 10 min each, the sections were incubated with a streptavidin–peroxidase reagent in a humidifying chamber at room temperature for 30 min. A freshly-prepared substrate/chromogen solution of 3,3¢-diaminobenzidine (DAB) in the provided buffer (mix of 5 lL concentrated DAB with 50 mL substrate 178
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buffer) was then used to visualize the antigen–antibody reaction. Finally, the sections were counterstained in Mayer’s hematoxylin. The sections were examined by conventional light microscopy (Leica microscope with image analyzer, Leica Qwin standard software; Leica Microsystems GmbH, Wetzlar, Germany). Assessment of immunohistochemically-stained sections Sections stained with the MIB-1 antibody were examined under conventional light microscopy. Cells were considered positive for the Ki-67 antigen if there was intranuclear DAB staining (brown). All stained nuclei were scored positive, regardless of the intensity of staining. Cells that lacked a clear nucleus were excluded. A minimum of 1000 cells was counted in each section. Cell counts were made at 400· magnification with a conventional light microscope. The number of positively-stained nuclei was expressed as a percentage of the total number counted. The Ki-67 LI was calculated from the ratio of the number of tumor cells stained by Ki-67 to the total number of tumor cells counted per section: Ki-67 LI ¼
Number of Ki-67-positive cells 100 : Total number of tumor cells observed
Actual proliferative activity was calculated by using the following equation: Actual proliferative activity ¼ Ki-67 or MIB-1 LI AgNOR count: The group mean for the Ki-67 LI and the actual proliferative activity was then derived. Statistical analysis The mean value and standard deviation of the Ki-67 LI and AgNOR count were calculated in each group. Differences among groups were assessed by one-way anova. Subsequent comparisons between groups were done using the Mann–Whitney U-test, and the Kruskal–Wallis test was used for comparisons between Bryne’s grades, AgNOR count, and Ki-67 LI. Spearman’s rank correlation was used to investigate the relationship between the Ki-67 LI and AgNOR count. The level of statistical significance was P < 0.05. Results The demographic details of the cases are shown in Table 1. The Ki-67 immunostaining observed as brown-colored nuclei in all of the groups is shown in Figure 1. AgNOR appear as dark brown or black intranuclear bodies against ª 2011 Blackwell Publishing Asia Pty Ltd
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Actual proliferating activity in carcinoma
Table 1. Demographic distributions of cases
Characteristics Age Mean SD Range Sex Male Female
NOM (n = 10)
Leukoplakia (n = 20)
OSCC (n = 20)
32.60 5.13 25–40
44.65 15.31 20–70
53.20 10.26 35–68
20 (100%) 0 (0%)
13 (65%) 7 (35%)
6 (60%) 4 (40%)
NOM, normal oral mucosa; OSCC, oral squamous cell carcinoma; SD, standard deviation.
a yellowish background in all the groups, as shown in Figure 2. There is significant variation of mean AgNOR counts, Ki-67 LI, and actual proliferation index (API) for normal oral mucosa (NOM), leukoplakia, and OSCC (P < 0.05) (Table 2). The mean AgNOR count per nucleus, Ki-67 LI, and API were significantly higher in OSCC than leukoplakia and NOM (P < 0.05). The mean AgNOR count per nucleus, Ki-67 LI, and API was signifi-
Figure 1. Photomicrograph of immunostaining for Ki-67. (a) In the normal oral mucosa, the Ki-67-positive cells are predominantly noted in the basal cells and few in the suprabasal cells of the epithelium. (b) In leukoplakia without dysplasia, the Ki-67-positive cells are found in the basal cell layer and suprabasal cell layer. (c) In leukoplakia without dysplasia, the Ki-67-positive cells are found in the basal cell layer, predominantly in suprabasal cell layers, and few in the prickle cell layer. (d) In welldifferentiated squamous cell carcinomas, the Ki-67-positive cells are noted as little to nil in central areas of cellular maturation and keratinization, to high reactivity in the more activelyproliferating peripheral area. (e) Ki-67-positive cells are noted in moderately differentiated squamous cell carcinomas. (f) Ki-67-positive cells are noted to be greatest in number in poorly differentiated squamous cell carcinomas (original magnification 400·).
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cantly higher in leukoplakia than NOM (P < 0.05). Leukoplakia with dysplasia showed a significantly higher Ki-67 LI and API, compared to leukoplakia without dysplasia, but the AgNOR count showed no statistically-significant difference (Table 3). The mean AgNOR count, mean Ki-67 LI, and mean API were significantly higher in OSCC than epithelial dysplasia (P < 0.05). There was no significant difference of mean AgNOR count, mean Ki-67 LI, and mean API among the different TNM stages of OSCC (Table 4). The mean values for AgNOR counts, Ki-67 LI, and API increased from well- to moderately- to poorlydifferentiated OSCC (Table 4). There was a significant correlation of Bryne’s histological malignancy grading with the AgNOR count and the Ki-67 LI (Table 4). There was significant positive correlation between the AgNOR count and the Ki-67 LI among all the groups (P < 0.05). Discussion NOM showed less positive cells for Ki-67, compared to that of oral leukoplakia and OSCC. For the leukoplakia without dysplasia, our results were in accordance with
(a)
(d)
(b)
(e)
(c)
(f)
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(a)
(d)
(b)
(e)
(c)
(f) Figure 2. Photomicrograph of argyrophilic nucleolar organizer region staining. (a) Normal oral mucosa; (b) leukoplakia without dysplasia; (c) leukoplakia with dysplasia; (d) welldifferentiated squamous cell carcinoma; (e) moderately differentiated squamous cell carcinoma; and (f) poorly differentiated squamous cell carcinoma (original magnification 1000·).
Table 2. Mean Ki-67 labeling index, AgNOR count per nucleus, and API in NOM, leukoplakia and OSCC
Group
n
Ki-67 labeling index mean (±SD)
NOM Leukoplakia OSCC
10 20 20
3.77 (±2.58) 20.50 (±13.16) 53.20 (±10.26)
AgNOR count/ nucleus mean (±SD)
API mean (±SD)
2.01 (±0.70) 2.55 (±0.59) 3.67 (±0.94)
7.41 (±5.08) 52.51 (±34.24) 180.90 (±87.76)
AgNOR, argyrophilic nucleolar organizer region; API, actual proliferative index; NOM, normal oral mucosa; OSCC, oral squamous cell carcinoma; SD, standard deviation.
other previous studies14–16 that found abrupt increases in the number of Ki-67-positive cells in the basal and parabasal layers of leukoplakia, as opposed to that in the normal epithelium. A significant increase of the Ki-67 LI was found in OSCC, compared to leukoplakia. This is in accordance with previous studies.17,18 Previous studies19–21 have emphasized the importance of recognizing proliferative activity beyond the basal layer, 180
Table 3. Mean AgNOR count, mean Ki-67 labeling index, and mean API between leukoplakia with dysplasia and leukoplakia without dysplasia
Leukoplakia
n
Ki-67 labeling index mean (±SD)
AgNOR count mean API mean (±SD) (±SD)
Leukoplakia 9 27.30 (±16.51) 2.73 (±0.49) with dysplasia Leukoplakia 11 14.90 (±5.86) 2.40 (±0.64) without dysplasia
71.91 (±39.42) 36.60 (±19.18)
AgNOR, argyrophilic nucleolar organizer region; API, actual proliferative index; SD, standard deviation.
and have highlighted the value of the suprabasal expression of Ki-67 as an objective marker of oral epithelial dysplasia that increases proportionately to the severity of dysplasia. In the present study, increased suprabasal Ki-67 expression was observed in severe dysplasia and OSCC. Our study showed a higher Ki-67 LI in leukoplakia with dysplasia, compared to leukoplakia without dysplasia. ª 2011 Blackwell Publishing Asia Pty Ltd
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Table 4. Mean AgNOR count, mean Ki-67 labeling index, and mean API, with clinical TNM staging and histological grading of OSCC
Actual proliferating activity in carcinoma
OSCC Clinical TNM stage Stage II Stage III Stage IV Histological stage WDSCC MDSCC PDSCC Bryne’s histological Good Moderate Poor
n
Ki-67 labeling index mean (±SD)
AgNOR count mean (±SD)
API mean (±SD)
1 8 11
3.17 (±0.0) 3.65 (±1.01) 3.72 (±0.96)
44.6 (±0.0) 43.2 (±14.90) 52.7 (±18.70)
141.38 (±0.0) 157.6 (±69.90) 201.41 (±100.78)
13 6 1 grading 2 13 5
3.49 (±0.85) 3.91 (±1.15) 4.49 (±0.0)
40.2 (±13.10) 62.53 (±12.31) 73.0 (±0.0)
140.49 (±55.40) 243.99 (±92.51) 327.77 (±0.0)
2.59 (±0.49) 3.72 (±0.75) 3.98 (±1.28)
26.94 (±4.32) 45.90 (±14.90) 64.04 (±12.20)
67.08 (±2.38) 170.33 (±61.07) 253.9 (±97.90)
AgNOR, argyrophilic nucleolar organizer region; API, actual proliferative index; MDSCC, moderately differentiated squamous cell carcinoma; OSCC, oral squamous cell carcinoma; WDSCC, well-differentiated squamous cell carcinoma; PDSCC, well-differentiated squamous cell carcinoma; SD, standard deviation.
In this study, a significant increase in the mean Ki-67 LI was noted from well-differentiated to moderatelydifferentiated OSCC. Carlos de Vicente et al.22 observed that there was a significant correlation between the Ki-67 LI and histological grading, while Piattelli et al.20 failed to show significant differences among well-, moderately-, and poorly-differentiated OSCC. These conflicting results might be due to disparity in the methodology, quantitation, and small sample size. In Bryne’s histological malignancy grading system, the Ki-67 LI was minimal in the good-prognosis group and higher in the poor-prognosis group, with an intermediate value in moderate group. However, we emphasize that caution is required when interpreting the results, as there were only two cases in the group representing good prognosis, and only one case of poorly-differentiated OSCC. It is clear from various studies22–24 and our study that high Ki-67 LI were observed in OSCC, which correlate with disease progression. The distribution of Ki-67positive cells varies from little to nil in central areas of cellular maturation and keratinization, to high reactivity in the more actively-proliferating peripheral portions. Our results showed no significant correlation with TNM staging, but there was a significant correlation of the Ki-67 LI with the WHO grading and Bryne’s histological malignancy grading. In this study, a significant increase in mean AgNOR counts was observed from NOM to non-dysplastic leukoplakia, to dysplastic leukoplakia and OSCC. Several previous study findings25,26 are in agreement with these findings. A quantitative analysis of the interphase nucleolar organizer region has proven to be valuable in tumor pathology for distinguishing malignant lesions from benign lesions of the same origin.27 Although these differª 2011 Blackwell Publishing Asia Pty Ltd
ences were significant, counts in each diagnostic group overlapped so much that they were of no practical value in distinguishing between individual lesions. AgNOR in normal epithelial cells appeared to be of regular size with a round shape, while in malignant tissue, they appeared less uniform in size and shape, and were often of unusual forms. Dysplastic epithelia showed mixed patterns. This feature was also observed by several other authors.28,29 There was no correlation between the AgNOR count and TNM staging. However, it was higher in stage IV followed by stage III and stage II. Hirsch et al.30 and Yue et al.26 reported a positive correlation between the AgNOR count and TNM staging, whereas another study31 showed no correlation. In the present study, a non-significant correlation of AgNOR counts was found among different histological grades. However, AgNOR counts showed higher value in poorly- and moderately-differentiated OSCC when compared with well-differentiated OSCC. Literature yields a sole report of no correlation;30 however, a positive correlation between AgNOR counts and histological grades was reported by most investigators.25,26,28 This might be because of an inadequate distribution of various categories, as our study included only one case of poorly-differentiated OSCC. In our study, AgNOR counts were highest in the poor-prognosis group, followed by the moderate- and good-prognosis groups for Bryne’s histological malignancy grading. The findings of most previous reports25,26,28 support our finding of a higher AgNOR count in the poor-prognosis group. Our results reaffirm the finding of previous studies,32,33 that is, an increase in AgNOR counts in dysplastic leukoplakia, compared with non-dysplastic leukoplakia and NOM. Thus, the AgNOR might be quantitative, 181
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discriminative, and easy to monitor routinely in detecting incipient cellular alterations.34 In our study, the mean AgNOR count for leukoplakia with dysplasia was 2.73. Longitudinal studies that take into consideration not only the number and distribution but also the area and volume of AgNOR using computerized and standardized techniques should be carried out to find the real prognostic use of the AgNOR in premalignant and malignant oral cavity lesions. Our study further observed a progressive increase in actual proliferative activity from NOM, followed by nondysplastic leukoplakia and dysplastic leukoplakia to OSCC. A significant correlation of the API was noted with the histological malignancy grading system. When applied in relation to Bryne’s grading, the API appeared to increase with worsening prognosis. However, we could not draw a statistically-significant correlation of the API with clinical staging using the TNM classification due to an inadequate sample size. Our study also demonstrated a significant positive correlation between the AgNOR count and the Ki-67 LI in all the groups. This suggests that the increased proliferative state and turnover rate of epithelial
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cells is correlated with the different biological behavior of epithelia in NOM, leukoplakia, leukoplakia with dysplasia, and OSCC. Considering the malignant potential of dysplastic leukoplakia, it is important not to miss a dysplastic case. The assessment of cell proliferation can cause a falsenegative diagnosis of dysplastic premalignant lesions if assessed solely on the basis of routine hematoxylin–eosin staining. Thus, it is suggested that tissues of leukoplakia or suspected epithelial dysplasia should be additionally stained for AgNOR and Ki-67, because using cell proliferation as a diagnostic criterion has a stronger biological basis. Multiple, larger studies will standardize the use of actual proliferative activity to distinguish between nondysplastic leukoplakia and dysplastic leukoplakia. Clinical applications of studies on cell proliferation have a long way to go. It is postulated that pharmacological modulators of cell proliferation and differentiation are the future of cancer-preventive drugs. Thus, the use of Ki-67 and AgNOR could be of great value in monitoring the effects of these agents during the course of future chemoprevention trials.
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ORIGINAL ARTICLE Oral Pathology
Actual proliferating index in oral squamous cell carcinoma and leukoplakia Abhay R. Chandak1, Amol Ramchandra Gadbail2, Minal S. Chaudhary2, Shweta A. Chandak3 & Ritesh Wadhwani4 1 Department of Oral and Maxillofacial Pathology and Microbiology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India 2 Department of Oral and Maxillofacial Pathology and Microbiology, Datta Meghe Institute of Medical Sciences, Sharad Pawar Dental College and Hospital, Sawangi (Meghe), Wardha, Maharashtra, India 3 Department of Pediatric and Preventive Dentistry, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India 4 Department of Oral and Maxillofacial Pathology and Microbiology, Jamanlal Goenka Dental College and Hospital, Akola, Maharashtra, India
Keywords actual proliferation index, argyrophilic nucleolar organizer region count, Ki-67 labeling index, oral premalignant lesion, oral squamous cell carcinoma. Correspondence Dr Amol Ramchandra Gadbail, Department of Oral and Maxillofacial Pathology and Microbiology, Datta Meghe Institute of Medical Sciences, Sharad Pawar Dental College and Hospital, Sawangi (Meghe), Wardha 442001, Maharashtra, India. Tel: +91-97661-35691 Fax: +91-071-5228-7713 Email: [email protected] Received 27 December 2010; accepted 29 January 2011. doi: 10.1111/j.2041-1626.2011.00057.x
Abstract Aim: To examine the possible association between epithelial proliferation and disease progression in the oral mucosa using the actual proliferation index. Methods: The actual proliferation index was measured by the Ki-67 labeling index and argyrophilic nucleolar organizer region count per nucleus. Immunohistochemistry was carried out for Ki-67 by using the molecular immunology borstel-1 clone in 20 leukoplakias, 20 oral squamous cell carcinomas, and 10 normal oral mucosae. Results: The argyrophilic nucleolar organizer region count per nucleus, Ki-67 labeling index, and actual proliferation index were significantly higher in oral squamous cell carcinoma, followed by leukoplakia and normal oral mucosa. Leukoplakia with dysplasia showed a significantly higher Ki-67 labeling index and actual proliferation index, compared to leukoplakia without dysphasia. There was a significant correlation of Bryne’s histological malignancy grading with the argyrophilic nucleolar organizer region count and the Ki-67 labeling index. There was a significant positive correlation between the argyrophilic nucleolar organizer region count and the Ki-67 labeling index among all groups. Conclusions: Leukoplakia or suspected epithelial dysplasia should be stained for argyrophilic nucleolar organizer regions and Ki-67. The actual proliferation index is not only useful as a prognostic factor, but could also be a promising treatment determining modality for patients with premalignant and malignant lesions.
Introduction Oral cancer holds eighth position in cancer incidence worldwide. A geographic variation in the incidence of oral squamous cell carcinoma (OSCC) exists. In India, OSCC ranks first among all cancers in males, and third in females. Incidence rates vary from seven to 17/100 000 people annually. Based on cancer registry data, it is estimated that 75 000–80 000 new cancer cases develop annually in India.1 176
Squamous cell carcinoma is the most common malignant neoplasm of the oral cavity, accounting for more than 90% of all oral malignancies. It is usually preceded by premalignant lesions. The identification of these premalignant lesions through clinical, morphological, and molecular means helps in the early detection and treatment of OSCC. Leukoplakia is the most common oral premalignant lesion.2 Oral premalignant lesions are known to represent an increased risk of oral cancer development, with quoted malignant transformation rates ª 2011 Blackwell Publishing Asia Pty Ltd
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varying from 6.6% to 36%.3 Changes in the proliferative capacity of oral premalignant lesions might indicate early neoplastic changes and the risk of malignant transformation.4 It is generally agreed that lesions exhibiting oral epithelial dysplasia are at increased risk of malignant transformation. Epithelial dysplasia is characterized by a number of cellular and tissue alterations that reveal an alteration of cell maturation in the epithelium. However, it has been noted that there might be subjective variation in the diagnosis and grade of the same sample of epithelial dysplasia by different pathologists. Therefore, markers of cellular proliferation might help to establish more objective diagnostic criteria. Previous studies on the proliferative characteristics of oral epithelia of precancer and cancer have been performed by using various methods.5,6 Among those most often used, immunohistochemical staining using the molecular immunology borstel-1 (MIB-1) antibody against the Ki-67 antigen has emerged as a marker of choice. In terms of cell biology, the proliferative state reflects the percentage of a proliferation-associated population at any particular time.7 The Ki-67 expression assesses the state of cell proliferation, that is, growth fraction (the proportion of cells committed to the cell cycle). The Ki-67 human nuclear antigen is expressed during the G1, S, G2, and M phases in the cell cycle, but is absent in the quiescent G0 phase.8 The proliferation rate represents measurements in which a cell-cycle phase transit time is calculated to allow the estimation of a doubling time.7 This can be evaluated by an argyrophilic nucleolar organizer region (AgNOR) parameter. Interphase AgNOR are structural and functional units of the nucleolus, in which all of the components necessary for ribosomal RNA synthesis are located. The number of interphase AgNOR are strictly related to rRNA transcriptional activity, and in continuously-proliferating cells, to the rapidity of cell proliferation.9 Therefore, the actual proliferative activity of a lesion can simply be expressed as: Actual proliferative activity = Ki-67 or MIB-1 scores · AgNOR quantity.6,10 In the present study, we aimed to examine the possible association between actual proliferative activity and disease progression in the oral mucosa using the Ki-67 labeling index (LI) and AgNOR count to determine true proliferative index. We further compare the clinical parameters and histological grading of OSCC in the context of cell proliferation. Methods The present study was carried out at the Department of Oral Pathology and Microbiology, Sharad Pawar Dental College, Wardha, India. The study included 20 histopathologically diagnosed OSCC patients, 20 oral leukoplakia ª 2011 Blackwell Publishing Asia Pty Ltd
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patients, and 10 normal oral mucosa patients. In the study, patients with oral leukoplakia had white and/or red lesions with no other etiological factor, except a habit of tobacco smoking, as well as tobacco quid and betel nut chewing. The clinical information was recorded for OSCC and leukoplakia following a definite format. Written consent was obtained from all patients. Approval for the study was given by the Institutional Ethical Committee of Datta Meghe Institute of Medical Sciences, Sawangi (Meghe), Wardha, India. The study was performed on tissues fixed in 10% neutral buffered formalin, paraffinembedded tissue. The sections were cut serially to a 4-lm thickness for AgNOR staining, and 5 lm for immunohistochemistry to evaluate the expression of the Ki-67 antigen. Leukoplakia samples were divided into two groups: with and without dysplasia. In OSCC, clinical TNM staging and histological malignancy grading (World Health Organization [WHO] and Bryne’s) was carried out. Bryne’s histological malignancy grading was performed on most anaplastic fields, and cases were separated into three categories, which had significantly different prognostic groups, as suggested by Bryne et al.11 Procedure AgNOR staining method Solution A Two grams (2% w/v) of gelatin powder was added to 100 mL distilled water, with the temperature maintained at 60–70C over a water bath. Later, 1 mL formic acid (1%) (H.COOH = 46.03; Qualigens Fine Chemicals, Mumbai, India) was added and the mixture was stirred thoroughly. Solution B Twenty-five grams of silver nitrate (AgNO3 = 169.87; Qualigens Fine Chemicals) was added to 50 mL distilled water. After the dissolution, the liquid was filtered with #1 Whatman filter paper (Qualigens Fine Chemicals). Working solution Twenty-five milliliters of solution A was mixed with 50 mL of solution B to obtain a resultant 75 mL of working solution. Staining was carried out according to the International Committee on AgNOR Quantitation.12 Slides were hydrated and heated in a 750-W microwave oven for 10 min in 10 mm sodium citrate buffer (pH 6.0). The sections were then washed three times with double-distilled water for 5 min each, and incubated in the dark for 20–25 min in a working solution at room temperature. The slides were washed in running doubledistilled water for 10–15 min. 177
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Quantification of AgNOR All the slides were examined by light microscopy at a 1000· magnification. In each section, a minimum of 200 nuclei were examined, and the mean number of AgNOR per cell was calculated. Counting was done according to the criteria of Giri et al.13 By careful focusing, clearlydefined dots of AgNOR within nucleoli and small AgNOR outside nucleoli were counted. AgNOR appears as dark brown or black intranuclear bodies against a yellowish background. When AgNOR were found lying in clusters and were not individually resolvable, the cluster of AgNOR was counted as a single AgNOR. Lymphocytes in the lamina propria were used as an internal control. For normal epithelia and leukoplakia, basal and adjacent parabasal layers were evaluated. In well-differentiated squamous cell carcinomas, keratinized cells were avoided because nuclear pyknosis obscure the AgNOR. Mean AgNOR per cell were calculated for each case, and group means were derived. Immunohistochemistry for detecting the Ki-67 antigen For immunohistochemistry, the peroxidase detection system (streptavidin–biotin detection system; Novocastra kit; Novocastra Laborotories, Newcastle-upon-Tyne, UK) was employed. The sections were deparaffinized with xylene and hydrated. The slides were heated in microwave oven for 10 min in 0.01 m sodium citrate buffer (pH 6.0) for antigen retrieval and bench cooled for 20 min; the cycle was then repeated. Endogenous peroxidase activity was blocked by incubating the section with 3% H2O2 in methanol for 30 min. The sections were then washed three times with gentle shaking in phosphate-buffered saline (PBS) for 5 min each. To prevent non-specific reactions, the sections were incubated with 10% serum for 10 min. Prediluted Ki-67 antibody (clone MIB-1; Dako, Glostrup, Denmark) was incubated at room temperature in a humidifying chamber for 60 min, and then at 4C overnight. The hyperplastic lymph node was used as a positive control. One section from the positive control was used as the negative control by omitting the primary antibody and by incubating with the serum. After the primary antibody and antigen reactions, the sections were rinsed in PBS three times for 10 min each. The secondary antibody (biotinylated rabbit antimouse immunoglobulin G) was incubated at room temperature in a humidifying chamber for 30 min. After washing the sections three times in PBS for 10 min each, the sections were incubated with a streptavidin–peroxidase reagent in a humidifying chamber at room temperature for 30 min. A freshly-prepared substrate/chromogen solution of 3,3¢-diaminobenzidine (DAB) in the provided buffer (mix of 5 lL concentrated DAB with 50 mL substrate 178
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buffer) was then used to visualize the antigen–antibody reaction. Finally, the sections were counterstained in Mayer’s hematoxylin. The sections were examined by conventional light microscopy (Leica microscope with image analyzer, Leica Qwin standard software; Leica Microsystems GmbH, Wetzlar, Germany). Assessment of immunohistochemically-stained sections Sections stained with the MIB-1 antibody were examined under conventional light microscopy. Cells were considered positive for the Ki-67 antigen if there was intranuclear DAB staining (brown). All stained nuclei were scored positive, regardless of the intensity of staining. Cells that lacked a clear nucleus were excluded. A minimum of 1000 cells was counted in each section. Cell counts were made at 400· magnification with a conventional light microscope. The number of positively-stained nuclei was expressed as a percentage of the total number counted. The Ki-67 LI was calculated from the ratio of the number of tumor cells stained by Ki-67 to the total number of tumor cells counted per section: Ki-67 LI ¼
Number of Ki-67-positive cells 100 : Total number of tumor cells observed
Actual proliferative activity was calculated by using the following equation: Actual proliferative activity ¼ Ki-67 or MIB-1 LI AgNOR count: The group mean for the Ki-67 LI and the actual proliferative activity was then derived. Statistical analysis The mean value and standard deviation of the Ki-67 LI and AgNOR count were calculated in each group. Differences among groups were assessed by one-way anova. Subsequent comparisons between groups were done using the Mann–Whitney U-test, and the Kruskal–Wallis test was used for comparisons between Bryne’s grades, AgNOR count, and Ki-67 LI. Spearman’s rank correlation was used to investigate the relationship between the Ki-67 LI and AgNOR count. The level of statistical significance was P < 0.05. Results The demographic details of the cases are shown in Table 1. The Ki-67 immunostaining observed as brown-colored nuclei in all of the groups is shown in Figure 1. AgNOR appear as dark brown or black intranuclear bodies against ª 2011 Blackwell Publishing Asia Pty Ltd
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Actual proliferating activity in carcinoma
Table 1. Demographic distributions of cases
Characteristics Age Mean SD Range Sex Male Female
NOM (n = 10)
Leukoplakia (n = 20)
OSCC (n = 20)
32.60 5.13 25–40
44.65 15.31 20–70
53.20 10.26 35–68
20 (100%) 0 (0%)
13 (65%) 7 (35%)
6 (60%) 4 (40%)
NOM, normal oral mucosa; OSCC, oral squamous cell carcinoma; SD, standard deviation.
a yellowish background in all the groups, as shown in Figure 2. There is significant variation of mean AgNOR counts, Ki-67 LI, and actual proliferation index (API) for normal oral mucosa (NOM), leukoplakia, and OSCC (P < 0.05) (Table 2). The mean AgNOR count per nucleus, Ki-67 LI, and API were significantly higher in OSCC than leukoplakia and NOM (P < 0.05). The mean AgNOR count per nucleus, Ki-67 LI, and API was signifi-
Figure 1. Photomicrograph of immunostaining for Ki-67. (a) In the normal oral mucosa, the Ki-67-positive cells are predominantly noted in the basal cells and few in the suprabasal cells of the epithelium. (b) In leukoplakia without dysplasia, the Ki-67-positive cells are found in the basal cell layer and suprabasal cell layer. (c) In leukoplakia without dysplasia, the Ki-67-positive cells are found in the basal cell layer, predominantly in suprabasal cell layers, and few in the prickle cell layer. (d) In welldifferentiated squamous cell carcinomas, the Ki-67-positive cells are noted as little to nil in central areas of cellular maturation and keratinization, to high reactivity in the more activelyproliferating peripheral area. (e) Ki-67-positive cells are noted in moderately differentiated squamous cell carcinomas. (f) Ki-67-positive cells are noted to be greatest in number in poorly differentiated squamous cell carcinomas (original magnification 400·).
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cantly higher in leukoplakia than NOM (P < 0.05). Leukoplakia with dysplasia showed a significantly higher Ki-67 LI and API, compared to leukoplakia without dysplasia, but the AgNOR count showed no statistically-significant difference (Table 3). The mean AgNOR count, mean Ki-67 LI, and mean API were significantly higher in OSCC than epithelial dysplasia (P < 0.05). There was no significant difference of mean AgNOR count, mean Ki-67 LI, and mean API among the different TNM stages of OSCC (Table 4). The mean values for AgNOR counts, Ki-67 LI, and API increased from well- to moderately- to poorlydifferentiated OSCC (Table 4). There was a significant correlation of Bryne’s histological malignancy grading with the AgNOR count and the Ki-67 LI (Table 4). There was significant positive correlation between the AgNOR count and the Ki-67 LI among all the groups (P < 0.05). Discussion NOM showed less positive cells for Ki-67, compared to that of oral leukoplakia and OSCC. For the leukoplakia without dysplasia, our results were in accordance with
(a)
(d)
(b)
(e)
(c)
(f)
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(a)
(d)
(b)
(e)
(c)
(f) Figure 2. Photomicrograph of argyrophilic nucleolar organizer region staining. (a) Normal oral mucosa; (b) leukoplakia without dysplasia; (c) leukoplakia with dysplasia; (d) welldifferentiated squamous cell carcinoma; (e) moderately differentiated squamous cell carcinoma; and (f) poorly differentiated squamous cell carcinoma (original magnification 1000·).
Table 2. Mean Ki-67 labeling index, AgNOR count per nucleus, and API in NOM, leukoplakia and OSCC
Group
n
Ki-67 labeling index mean (±SD)
NOM Leukoplakia OSCC
10 20 20
3.77 (±2.58) 20.50 (±13.16) 53.20 (±10.26)
AgNOR count/ nucleus mean (±SD)
API mean (±SD)
2.01 (±0.70) 2.55 (±0.59) 3.67 (±0.94)
7.41 (±5.08) 52.51 (±34.24) 180.90 (±87.76)
AgNOR, argyrophilic nucleolar organizer region; API, actual proliferative index; NOM, normal oral mucosa; OSCC, oral squamous cell carcinoma; SD, standard deviation.
other previous studies14–16 that found abrupt increases in the number of Ki-67-positive cells in the basal and parabasal layers of leukoplakia, as opposed to that in the normal epithelium. A significant increase of the Ki-67 LI was found in OSCC, compared to leukoplakia. This is in accordance with previous studies.17,18 Previous studies19–21 have emphasized the importance of recognizing proliferative activity beyond the basal layer, 180
Table 3. Mean AgNOR count, mean Ki-67 labeling index, and mean API between leukoplakia with dysplasia and leukoplakia without dysplasia
Leukoplakia
n
Ki-67 labeling index mean (±SD)
AgNOR count mean API mean (±SD) (±SD)
Leukoplakia 9 27.30 (±16.51) 2.73 (±0.49) with dysplasia Leukoplakia 11 14.90 (±5.86) 2.40 (±0.64) without dysplasia
71.91 (±39.42) 36.60 (±19.18)
AgNOR, argyrophilic nucleolar organizer region; API, actual proliferative index; SD, standard deviation.
and have highlighted the value of the suprabasal expression of Ki-67 as an objective marker of oral epithelial dysplasia that increases proportionately to the severity of dysplasia. In the present study, increased suprabasal Ki-67 expression was observed in severe dysplasia and OSCC. Our study showed a higher Ki-67 LI in leukoplakia with dysplasia, compared to leukoplakia without dysplasia. ª 2011 Blackwell Publishing Asia Pty Ltd
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Table 4. Mean AgNOR count, mean Ki-67 labeling index, and mean API, with clinical TNM staging and histological grading of OSCC
Actual proliferating activity in carcinoma
OSCC Clinical TNM stage Stage II Stage III Stage IV Histological stage WDSCC MDSCC PDSCC Bryne’s histological Good Moderate Poor
n
Ki-67 labeling index mean (±SD)
AgNOR count mean (±SD)
API mean (±SD)
1 8 11
3.17 (±0.0) 3.65 (±1.01) 3.72 (±0.96)
44.6 (±0.0) 43.2 (±14.90) 52.7 (±18.70)
141.38 (±0.0) 157.6 (±69.90) 201.41 (±100.78)
13 6 1 grading 2 13 5
3.49 (±0.85) 3.91 (±1.15) 4.49 (±0.0)
40.2 (±13.10) 62.53 (±12.31) 73.0 (±0.0)
140.49 (±55.40) 243.99 (±92.51) 327.77 (±0.0)
2.59 (±0.49) 3.72 (±0.75) 3.98 (±1.28)
26.94 (±4.32) 45.90 (±14.90) 64.04 (±12.20)
67.08 (±2.38) 170.33 (±61.07) 253.9 (±97.90)
AgNOR, argyrophilic nucleolar organizer region; API, actual proliferative index; MDSCC, moderately differentiated squamous cell carcinoma; OSCC, oral squamous cell carcinoma; WDSCC, well-differentiated squamous cell carcinoma; PDSCC, well-differentiated squamous cell carcinoma; SD, standard deviation.
In this study, a significant increase in the mean Ki-67 LI was noted from well-differentiated to moderatelydifferentiated OSCC. Carlos de Vicente et al.22 observed that there was a significant correlation between the Ki-67 LI and histological grading, while Piattelli et al.20 failed to show significant differences among well-, moderately-, and poorly-differentiated OSCC. These conflicting results might be due to disparity in the methodology, quantitation, and small sample size. In Bryne’s histological malignancy grading system, the Ki-67 LI was minimal in the good-prognosis group and higher in the poor-prognosis group, with an intermediate value in moderate group. However, we emphasize that caution is required when interpreting the results, as there were only two cases in the group representing good prognosis, and only one case of poorly-differentiated OSCC. It is clear from various studies22–24 and our study that high Ki-67 LI were observed in OSCC, which correlate with disease progression. The distribution of Ki-67positive cells varies from little to nil in central areas of cellular maturation and keratinization, to high reactivity in the more actively-proliferating peripheral portions. Our results showed no significant correlation with TNM staging, but there was a significant correlation of the Ki-67 LI with the WHO grading and Bryne’s histological malignancy grading. In this study, a significant increase in mean AgNOR counts was observed from NOM to non-dysplastic leukoplakia, to dysplastic leukoplakia and OSCC. Several previous study findings25,26 are in agreement with these findings. A quantitative analysis of the interphase nucleolar organizer region has proven to be valuable in tumor pathology for distinguishing malignant lesions from benign lesions of the same origin.27 Although these differª 2011 Blackwell Publishing Asia Pty Ltd
ences were significant, counts in each diagnostic group overlapped so much that they were of no practical value in distinguishing between individual lesions. AgNOR in normal epithelial cells appeared to be of regular size with a round shape, while in malignant tissue, they appeared less uniform in size and shape, and were often of unusual forms. Dysplastic epithelia showed mixed patterns. This feature was also observed by several other authors.28,29 There was no correlation between the AgNOR count and TNM staging. However, it was higher in stage IV followed by stage III and stage II. Hirsch et al.30 and Yue et al.26 reported a positive correlation between the AgNOR count and TNM staging, whereas another study31 showed no correlation. In the present study, a non-significant correlation of AgNOR counts was found among different histological grades. However, AgNOR counts showed higher value in poorly- and moderately-differentiated OSCC when compared with well-differentiated OSCC. Literature yields a sole report of no correlation;30 however, a positive correlation between AgNOR counts and histological grades was reported by most investigators.25,26,28 This might be because of an inadequate distribution of various categories, as our study included only one case of poorly-differentiated OSCC. In our study, AgNOR counts were highest in the poor-prognosis group, followed by the moderate- and good-prognosis groups for Bryne’s histological malignancy grading. The findings of most previous reports25,26,28 support our finding of a higher AgNOR count in the poor-prognosis group. Our results reaffirm the finding of previous studies,32,33 that is, an increase in AgNOR counts in dysplastic leukoplakia, compared with non-dysplastic leukoplakia and NOM. Thus, the AgNOR might be quantitative, 181
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discriminative, and easy to monitor routinely in detecting incipient cellular alterations.34 In our study, the mean AgNOR count for leukoplakia with dysplasia was 2.73. Longitudinal studies that take into consideration not only the number and distribution but also the area and volume of AgNOR using computerized and standardized techniques should be carried out to find the real prognostic use of the AgNOR in premalignant and malignant oral cavity lesions. Our study further observed a progressive increase in actual proliferative activity from NOM, followed by nondysplastic leukoplakia and dysplastic leukoplakia to OSCC. A significant correlation of the API was noted with the histological malignancy grading system. When applied in relation to Bryne’s grading, the API appeared to increase with worsening prognosis. However, we could not draw a statistically-significant correlation of the API with clinical staging using the TNM classification due to an inadequate sample size. Our study also demonstrated a significant positive correlation between the AgNOR count and the Ki-67 LI in all the groups. This suggests that the increased proliferative state and turnover rate of epithelial
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