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Ki-67, CD105, and α-smooth muscle actin expression in oral squamous cell carcinoma corresponds with different forms of tobacco consumption habits


1 Department of Dentistry, Indira Gandhi Government Medical College and Hospital, Nagpur, Maharashtra, India
2 Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Dental College and Hospital, Dr. D.Y. Patil Vidyapeeth, Pune, Maharashtra, India
3 Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences, Wardha, Maharashtra, India
4 Department of Oral Medicine and Radiology, Government Dental College and Hospital, Nagpur, Maharashtra, India
5 Department of Oral Pathology and Microbiology, Modern Dental College and Research Centre, Indore, Maharashtra, India
6 Department of Oral Pathology and Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India
7 Department of Maxillofacial Surgery and Diagnostic Sciences, Division of Oral Pathology, College of Dentistry, Jazan University, Jazan, Saudi Arabia

Date of Submission05-Sep-2020
Date of Decision20-Dec-2020
Date of Acceptance22-Jan-2021
Date of Web Publication25-Oct-2021

Correspondence Address:
Sachin C Sarode,
Department of Oral and Maxillofacial Pathology, Dr. D.Y.Patil Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune - 411 018, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_1307_20

 > Abstract 


Background: Association with variety of etiological agents is one of the characteristic features of oral squamous cell carcinoma (OSCC). We hypothesized the existence of tobacco consumption habit-based heterogeneity in the immunohistochemical expression of carcinogenesis relevant molecular markers in OSCC. Hence, the present study was conducted to investigate the carcinogenesis relevant three commonly expressed markers (Ki-67, CD105, and α-smooth muscle acting [SMA]) in various forms of tobacco consumption habits in OSCC patients.
Materials and Methods: A total of 217 patients of OSCC were included in the study, and based on the habit, they were broadly categorized into tobacco lime (TL), TL and areca nut (TLAN), and areca nut (AN). Further, categorization was done on the basis of absence or presence of additional habit of smoking. Immunohistochemistry (IHC) was performed using Ki-67, CD105, and α-SMA markers on formalin-fixed paraffin-embedded tissues.
Results: TLAN (62.21%) was the most common habit noted in OSCC patient followed by TL (20.73%) and AN (15.20%). The additional habit of smoking was observed in 31.11% and 25.92% of TL and TLAN habits of OSCC patients, respectively. All the three markers (Ki-67, CD105, and α-SMA) showed statistically significant differences in the habit group such as TL, TLAN, and AN (P < 0.001). Although the expression of all the three markers was increased in TL as compared with TLAN, differences were not statistically significant. When these markers were compared in with and without smoking category, only TLAN with smoking and TLAN without smoking showed statistically significant differences in the expression of all three markers.
Conclusions: Ki-67 CD105 and α-SMA immunohistochemical expression in OSCC corresponds with different forms of tobacco consumption habits. Habit-related unique carcinogenesis events are reflected at IHC level thus providing proof of concept for future studies.

Keywords: Areca nut, CD105, habit, Ki 67, oral squamous cell carcinoma, tobacco, α-smooth muscle acting



How to cite this URL:
Gadbail AR, Sarode SC, Chaudhary MS, Gondivkar SM, Tekade SA, Yuwanati M, Sarode GS, Hande A, Patil S. Ki-67, CD105, and α-smooth muscle actin expression in oral squamous cell carcinoma corresponds with different forms of tobacco consumption habits. J Can Res Ther [Epub ahead of print] [cited 2021 Nov 29]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=329187




 > Introduction Top


One of the unique features of oral squamous cell carcinoma (OSCC) is its association with varied nature of carcinogenic attack derived from smokeless tobacco, smoked tobacco, areca quid, alcohol, human papilloma virus, chronic trauma, and so on.[1] All the factors alone and/or in various permutation sand combinations produce wide-ranging carcinogenic attacks leading to molecular alteration, which could add to the already existing inter-tumoral heterogeneity.[2] In this regard, differences in the molecular alterations in OSCC of Western countries as compared to Eastern countries were ascribed to etiological and/or ethnic origin.[3] However, data on the habit-related variability of molecular alteration in OSCC are very scanty in the literature. High incidences of H-ras mutations (codons 12, 13 or61) were reported in OSCC in the Indian tobacco chewing population[4] in comparison with smokers from the industrialized Western populations.[5],[6] Moreover, the analysis of p53 mutations showed that G-A, C-T, and G-T mutations that are associated with tobacco-specific nitrosamines have been found to be the most common in smokeless tobacco-associated oral cancers.[7]

With respect to oral carcinogenesis, majority of the studies in the literature investigated the molecular mechanisms of carcinogenesis by considering the carcinogenic agent in isolation. It is paramount importance to consider the real life practice of chewing and smoking habits, which always presents in permutation and combinations. Due to changing trends in the tobacco and areca nut (AN) consumption pattern, it is conceivable to expect the wide variety of habit-based changes in carcinogenesis events.[8] Hence, we hypothesized the existence of habit-based heterogeneity in the immunohistochemical expression of carcinogenesis relevant molecular markers in OSCC. In the present study, we choose markers related to three important hallmarks of carcinogenesis, Ki-67 (proliferation), CD105 (angiogenesis), and α-smooth muscle acting (α-SMA) (invasion and metastasis). Ki-67 is a reliable maker for the proliferation and has been found to be well correlated with the biological behavior and prognosis of the various malignancies of the body including OSCC. Similarly, CD105 is known for its sensitivity and specificity for the newly formed blood vessels and hence represents true neo-angiogenesis. α-SMA is mostly regarded as the marker of epithelial-mesenchymal transition and thus is vital in predicting the local as well as distant metastasis.

With this view in mind and to establish the proof of concept, the present study was conducted to investigate the carcinogenesis relevant three commonly expressed markers (Ki-67, CD105, and α-SMA) in the various forms of tobacco consumption habits. This new objectives were investigated on the samples, which were utilized in our previously published papers.[9],[10]


 > Materials and Methods Top


The present study was carried out at the department of oral and maxillofacial pathology and microbiology at Sharad Pawar Dental College and Hospital, Wardha, Maharashtra, India. The ethical approval for this study was obtained from the Institutional Ethics Committee of Datta Meghe Institute of Medical Sciences, Wardha, Maharashtra India (Ref no. DMIMS (DU)/IEC/2014-15/953, dated 15/12/2014).

The study population was retrieved from the year 2010 to 2015, which was previously utilized for our research publications with different objectives.[9],[10] Patients with clinical and histopathological evidence of OSCC were included in the present study. Demographic data including detailed clinical presentation, detailed history of relevant habits along with duration of habits, and histopathological features were noted. A total of 217 patients of OSCC were included in the study. Based on the data retrieved, the natures of habits were broadly categorized into tobacco lime (TL), tobacco lime and AN (TLAN), and AN. This division is purely based on the composition of the nature of the tobacco consumption habit of the patients. The OSCC patients with habit of TL, TLAN, and AN were further categorized on the basis of absence or presence of addition habit of smoking as TL without smoking (TLWS), TLAN without smoking (TLAN-WS), areca nut without smoking (ANWS), TL with smoking (TLS), TLAN with smoking (TLAN-S), and AN with smoking. Neutral-buffered formalin fixed and paraffin-embedded tissue were retrieved from the departmental archive and used for immunohistochemistry (IHC).

Immunohistochemistry

The standard procedure of IHC was carried out for Ki-67, CD105, and α-SMA by using the appropriate controls on paraffin embedded tissue. Prediluted α-SMA antibody (Monoclonal Mouse Anti-Human [MMAH], Clone: 1A4; Product code [PC]: IR611, Dako, Denmark [DD]), prediluted Ki-67 antibody (clone MIB-1; PC: N1633; DD), and CD105 antibody (Diluted 1:30, MMAH, Clone: SN6 h, PC: M3527, DD) were used for IHC detection of Ki-67, CD105, and α-SMA antigen, respectively. The HRP-labeled polymer Anti-mouse secondary antibody (Dako EVision System, PC: K4000, DD.) was used as secondary antibody.

Immunohistochemistry scoring

Assessment of Ki-67-positive cells

In OSCC, neoplastic epithelial cells of invasive tumor front areas showing brown stained nuclei were considered as Ki-67 positive cells. The most heavily Ki-67-labeled areas were located by scanning the sections at × 100 magnification. Cell counts were made at × 400 magnification in five randomly selected fields. The number of positively stained nuclei was expressed as a percentage of the total number counted epithelial cells. Ki-67 labeling index (LI) = Number of IHC positive cells × 100/total number of cells observed.[11]

Assessment of CD105-positive cells

CD105-positive vascular endothelial cells were identified by their brown cytoplasmic staining. Areas with evidence of inflammation were avoided for the assessment of CD105 cells. Criteria given by Weidner et al.[12] 1991 were used for counting the microvessels. After scanning, the mean vascular density (MVD) was measured by counting CD-105-positive vessels in two hotspot areas at × 100 using Leica Qwin standard software and Leica DM LB 2 research microscope. The MVD was assessed in invasive tumor front area in OSCC.[11]

Assessment of alpha-smooth muscle acting positive cells

Irrespective of the intensity of staining, intra-cytoplasmic stained cells (other than noninflammatory and nonendothelial) with α-SMA were regarded as myofibroblasts. The percentage of cells positive for α-SMA in the tumor stroma was recorded as: 0 = No positive cells, 1 = 1%–33% positive cells, 2 = 34%–66% positive cells, and 3 = 67%–100% positive cells by three observers.[13] The same score obtained by more than two observers was counted as the final score. Scores 1, 2, and 3 were graded as mild, moderate, and intense expression of α-SMA, respectively. The expression of α-SMA was assessed in invasive tumor front area in OSCC.[11]

Statistical analysis

The data were statistically analyzed using SPSS, version 17.0 (IBM, New York, United States) for Windows. One-way analysis of variance and Tukey's HSD test were applied for the differences in Ki-67 LI, MVD, and α-SMA among the various habits groups in OSCC. The Mann–Whitney test was applied to find out the differences of Ki-67 LI, MVD, and α-SMA between smokers and nonsmokers of OSCC patient with habits of TL and TLAN. The level of statistical significance was at P < 0.05.


 > Results Top


Out of 217 OSCC patients, 159 (73.27%) were male and 35 (26.72%) were female. The mean age of OSCC patient was 50.25 (±12.14) years, and age ranges from 20 years to 79 years. The sites of OSCC, in nearly two-third patients were buccal mucosa (38.70%) and gingiva-buccal sulcus (31.79%). The other site for OSCC was tongue (11.05%), retromolar region (7.37%), labial mucosa (5.06%), palate (4.60%), and floor of mouth (1.38%). More than 50% of OSCC patients were belongs to lower socioeconomic status (64.97%), followed by middle (28.11%) and upper (28.11%). TLAN 135 (62.21%) was the most common habit noted in OSCC patient followed by TL 45 (20.73%) and AN 33 (15.20%). The additional habit of smoking was observed in 14 (31.11%) and 35 (25.92%) of TL and TLAN habits of OSCC patients. The additional habit of smoking was not observed in OSCC patients with habit of AN [Table 1].
Table 1: Details of demographic and clinicopathological parameters of oral squamous cell carcinoma patients included in the present study

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Duration of habit in oral squamous cell carcinoma

Statistically significant variations of mean of duration of habit were noted among all habits groups (P < 0.001). The higher duration of habit was observed in TL (30.17 [±10.95] years) followed by AN (25.21 [±11.96] years) and TLAN (22.94 [±10.31] years). Duration of habit was significantly higher with habit of TL as compared to TLAN. Nonsignificant difference of duration of habit was found between TL and AN and TLAN and AN.

The higher duration of habit was observed in TLWS (30.80 [±10.57] years) followed by TLS (28.78 [±12.04] years), ANWS (25.21 [±11.96] years), TLAN-WS (23.50 [±10.44] years), and TLAN-S (21.37 [±9.90] years). Duration of habit was significantly higher with habit of TLWS as compared to TLAN-WS (P = 0.013) and TLAN-S (P = 0.005) [Table 2].
Table 2: Details of various aspects of habits in oral squamous cell carcinoma patients

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Ki-67 labeling index and habits in oral squamous cell carcinoma

Statistically significant variations of mean Ki-67 LI noted among all habits groups (P < 0.001). Ki-67 LI were found significantly higher with habit of TL (56.89 [±12.25]) and TLAN (54.60 [±11.10]) as compared to AN (40.54 [±4.91]). Nonsignificant difference of Ki-67 LI was observed between TL and TLAN (P = 0.615). However, Ki-67 was higher in TL [Figure 1] and [Table 3].
Figure 1: Photomicrograph showing immune-expression in tumor cells and stroma oral squamous cell carcinoma of various habits groups: Ki-67 expression (KI-67 labeling index) in (a) tobacco lime (b) TLAN (c) areca nut; CD105 expression (mean vascular density) in (d) tobacco lime (e) TLAN (f) areca nut; and α-smooth muscle acting expression in (g) tobacco lime (h) TLAN (i) areca nut (Immunohistochemistry; Magnification ×100)

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Table 3: Comparison for Ki-67 LI, CD105 (mean vascular density) and α-smooth muscle actin expression in oral squamous cell carcinoma with respect to various tobacco and areca nut chewing habits

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In OSCC patients with habits of tobacco, lime, and AN (TLAN), Ki-67 LI was found significantly higher with smokers (TLAN-S) 60.03 (±11.92) as compared to without smokers (TLAN-WS) 52.70(±10.64) (P = 0.001) [Figure 2] and [Table 4].
Figure 2: Photomicrograph showing immune-expression in various habits groups of oral squamous cell carcinoma: Ki-67 expression (KI-67 labeling index) in neoplastic cells (a) TLAN-without smoking and (b) TLAN-S; and α-smooth muscle acting expression in tumor stroma (c) TLAN-without smoking and (d) TLAN-S (Immunohistochemistry; Magnification × 100)

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Table 4: Comparison for Ki-67 LI, CD105 (mean vascular density), and α-smooth muscle actin expression in oral squamous cell carcinoma with respect to tobacco chewing and smoking habit

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In OSCC patients with habits of TL, nonsignificant difference of Ki-67 LI was found between with smokers (TLS) and without smokers (TLWS) (P = 0.064). However, Ki-67 LI was higher in TLS (60.03 [±11.92]) than in TLWS (52.70 [±10.64]).

CD 105 and habit in oral squamous cell carcinoma

Statistically significant variations of mean MVD noted among all habits groups (P < 0.001). MVD was found significantly higher with habit of TL (85.15 [±14.50]) and TLAN (79.96 [±13.99]) as compared to habit of AN (62.78 [±7.89]). Nonsignificant difference of MVD was observed between habit of TL and TLAN (P = 0.114). However, MVD was higher in TL [Figure 1] and [Table 3].

In OSCC patients with habits of TLAN, nonsignificant difference of MVD was found between with smokers (TLAN-S) and without smokers (TLAN-WS) (P = 0.116). However, MVD was higher in TLAN-S (82.37 [±12.94]) than in TLAN-WS (79.12 [±14.30]).

In OSCC patients with habits of TL, nonsignificant difference of MVD was found between with smokers (TLS) and without smokers (TLWS) (P = 0.134). However, MVD was higher in TLS (88.35 [±11.16]) than in TLWS (83.70 [±15.73]).

Alpha-smooth muscle acting and habit in oral squamous cell carcinoma

Statistically significant variations of α-SMA expression were noted among all habits groups (P < 0.001). The α-SMA expression was significantly higher with habit of TL (2.15 [±0.92]) and TLAN (1.99 [±0.85]) as compared to habit of AN (1.03 [±0.76]) [Figure 1] and [Table 3]. Nonsignificant difference of α-SMA expression was observed between habit of TL and TLAN (P = 0.692). However, α-SMA expression was higher in habit of TL.

In OSCC patients with habits of TLAN, α-SMA expression was found significantly higher with smokers (TLAN-S) (2.28 [±0.78]) as compared to without smokers (TLAN-WS) (1.89 [±0.86]) (P = 0.016) [Figure 2] and [Table 4].

In OSCC patients with habits of TL, nonsignificant difference of α-SMA expression was found between with smokers (TLS) and without smokers (TLWS) (P = 0.281). However, α-SMA expression was higher in TLS (2.35 [±0.92]) than in (TLWS) (2.06 [±0.92]).


 > Discussion Top


One of the characteristic features of the OSCC is its association with the variety of causative agent including tobacco consumption.[14] There are different practices of tobacco consumption across the world with geographical variations. Smoking along with alcohol consumption is common in Europe, USA, Australia, China, and Japan, whereas smokeless tobacco practice is common in India, Sri Lanka, Papua New Guinea, and South-east Asia.[3] Smokeless tobacco consumption practice is highly diversified due to additions of other ingredients, especially AN and lime. We believe that each unique tobacco consumption habit, due to additives, harbors distinctive carcinogenesis mechanism and could be one the major factor responsible for the inter-tumoral heterogeneity. Literature is flooded with the studies on the molecular analysis of smokeless tobacco, smoked tobacco, and AN-associated carcinogenesis. However, these studies considered individual ingredient in isolation, which rarely happens in real life situation and thus promulgated the dire need for research in this direction. To address this contention, correlations of different forms of tobacco consumption habits with the immunohistochemical markers were presented as proof of concept. The categorization of the forms of tobacco consumption habits in the present study is purely based on the data obtained from the patients. As tobacco consumption pattern shows regional variation with various permutations and combinations, future studies are recommended in the different geographical location of world.

In the present study, TLAN was the most common habit noted in OSCC patient followed by TL and BN. The presence of higher number of patients in low socioeconomic group compliments these results. Based on personal observation, it was quite conceivable to expect the combination of smokeless tobacco and smoked tobacco habit in a single patients. Accordingly, history of smoking was reported in 14 (31.11%) and 35 (25.92%) of TL and TLAN habits of OSCC patients, respectively. Only BN chewing habit was less predominant and was mainly associated with the female population. These results are in accordance with the previously published literature on the Indian population.[15],[16]

Ki-67 is a nuclear and nucleolar protein, which is expressed in cell cycle from the G1 to the M phase and not in G0 phase.[17] Ki-67 is one of the best markers to identify the proliferating cells in tumor.[18] Ki-67 expression is associated with tumor cells proliferation and invasion and thus providing a marker of tumor aggressiveness.[19] In the present study, Ki-67 LI was found significantly higher with habit of TL and TLAN as compared to AN. Although Ki-67 LI was slightly higher in TL than TLAN, there difference was not statistically significant. Higher values of Ki-67 in TL group indicate that this pattern of habit could have up-regulating impact on the proliferation-related signaling cascades. Many signaling molecules, especially growth factors, have to impact the cell cycle in order for proliferation to successfully proceed. The pathways predicted to be upregulated in TL group could be AKT signaling and the TNF superfamily pathways. Moreover, mitogenic and anti-proliferative signals exert their effects on cell proliferation through the transcriptional regulation and ubiquitin-dependent degradation of cyclins and CDK inhibitors. On the other hand, AN-associated carcinogenesis might not be efficient driver of proliferation. Hence, OSCC associated with oral submucous fibrosis is majorly associated with better grade of tumor differentiation and good prognosis. Due to higher Ki-67 LI in TLAN-S as compared to TLAN-WS, smoking could be regarded as complimenting the smokeless tobacco carcinogenesis. However, nonsignificant difference of Ki-67 LI between TLS and TLWS refute the above contention.

Neoangiogenesis is an important step in tumor progression. Neoangiogenesis is determined by measuring MVD in given tissue. The measurement of MVD is aided in assessing tumor metastasis, recurrence, or survival.[20] CD105 is a hypoxia-induced protein and more specific marker in the evaluation of tumor neoangiogenesis.[21] Statistically significant variations of mean MVD were noted among all habits groups suggesting habit-related discriminatory effect on angiogenesis. MVD was found significantly higher with habit of TL and TLAN as compared to habit of AN. Apart from carcinogenic events, AN is also known to cause fibrosis by virtue of upregulation of transforming growth factor-beta mediated signaling pathways. We believe that decreased MVD count in AN group could be attributed to this fibrogenic mechanism of action. Intriguingly, there were no significant differences between the TL and TLAN suggesting dominating effect of tobacco in the upregulation of angiogenesis relevant biological process, which overpowers the fibrogenic potential of AN. However, smoking history could not able to differentiate between the TLS and TLWS groups as well as between TLAN-S and TLAN-WS.

The cancer-associated fibroblasts are activated fibroblast, which play multiple roles in promoting tumor growth, invasion, and metastasis. The cancer-associated fibroblasts are characterized by expression of α-SMA protein.[22] The cancer-associated fibroblast would have potential to induce an aggressive tumor phenotype by autocrine and paracrine expression of various growth factors, cytokines, and several proteolytic enzymes. The α-SMA expression helps in the prediction of invasive behavior, local recurrence, and survival of carcinoma.[23],[24] In the present study, α-SMA expression was significantly different in all the habit groups. Comparable to Ki-67 LI and MVD, α-SMA expression was also significantly higher with habit of TL and TLAN as compared to BN chewing habit, thus further strengthening the proof of concept proposed for the present study. Tobacco in TL and TLAN habit pattern could be associated with invasive nature, metastatic potential, and poorer prognosis of OSCC. However, insignificant differences of α-SMA expression between TL and TLAN might be suggestive of homogeneity in the α-SMA-mediated carcinogenic events in the two groups. Although smoking has significant promoting effect on α-SMA with TLAN-S, the results were insignificant for TLS and TLWS. The unknown intervening effect of AN could be possible reasoning for such discrimination, which needs further exploration in future.

In the present study, inclusion of only limited tobacco consumption habit-related groups was the inadvertent limitation, which prevented us from doing more comprehensive habit-marker correlation to establish proof of concept. Alcohol has been regarded as one of the key synergistic agents in the initiation and progression of oral carcinogenesis and has been shown to modulated key signaling pathways. Hence, alcohol is potential candidate for affecting the immune expression of the selected makers. Consideration of alcohol habit for correlation with biological marker is highly recommended in future studies. The present study has included that only three biological markers for investigations are another major limitation. However, we believe that these three markers represent the cardinal events in carcinogenesis and hence are enough to establish the proposed proof of concept.


 > Conclusions Top


Ki-67 CD105 and α-SMA immune-expression in OSCC showed statistically significant differences among different forms of tobacco consumption habits except between TN and TNAN. Thus, tobacco consumption habit-related unique carcinogenesis events are reflected in immune-expression level thus providing the proof of concept for future studies. Tobacco-lime group showed increased expression of Ki-67, MVD and alpha-SMA followed by tobacco-lime- AN group and AN group. These results suggest that tobacco (smokeless and smoking) could be potent in contemplating carcinogenesis relevant biological processes, which needs authentication in future molecular investigations. We recommend that future molecular investigations on pathogenesis should take into consideration the complete tobacco consumption habit pattern rather than individual components of the habit. These molecular investigations could involve genomic, epigenomic, proteomic and metabolomics exploration of various signaling events, and their associated molecules. Correlation of these explorations with the habit pattern will also help unraveling the intra and inter-tumoral heterogeneity attributed to diversified carcinogenic attacks in OSCC. This knowledge would be helpful in the development of personalized cancer medicine in future, which is heavily governed by the inter-tumoral and intra-tumoral heterogeneity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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