|Year : 2015 | Volume
| Issue : 1 | Page : 18-24
Evaluation of histomorphological and immunohistochemical parameters as biomarkers of cervical lymph node metastasis in squamous cell carcinoma of oral cavity: A retrospective study
TN Suresh1, A Hemalatha1, ML Harendra Kumar1, SM Azeem Mohiyuddin2
1 Department of Pathology, Sri Devaraj Urs Medical College, Sri Devaraj Urs Academy of Higher Education and Research Center, Kolar, Karnataka, India
2 Department of Ear, Nose and Throat, Head and Neck Surgery, Sri Devaraj Urs Medical College, Sri Devaraj Urs Academy of Higher Education and Research Center, Kolar, Karnataka, India
|Date of Submission||28-Jan-2014|
|Date of Acceptance||01-Apr-2015|
|Date of Web Publication||19-May-2015|
Dr. T N Suresh
Department of Pathology, Sri Devaraj Urs Medical Colege, Sri Devaj Urs Academy of Higher Education and Research Centre, Kolar - 562 103, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Oral squamous cell carcinoma (OSCC) is seen worldwide but is more common in India. Lymph node (LN) metastasis has been shown to be the strongest prognostic factor in OSCC. Many histopathological and immunohistochemical markers have been studied to predict the LN metastasis. Aims: To identify clinicopathological factors and immunohistochemical (IHC) biomarkers which predict cervical metastasis in OSCC patients. Study and Design: A total of 105 cases of OSCC were taken up for our study. Histopathological parameters such as tumor thickness, depth, degree of differentiation, pattern of invasion (POI), lymphovascular and neural invasion were assessed. IHC was done on all cases using antibodies against Ki-67, cyclin D, E-cadherin, p53, CD31 and each antibody was assessed according to the standard protocol. Statistical Analysis: To calculate the relation between clinical, histopathological parameter, IHC marker and the occurrence of LN metastasis, chi-square test was used. Variables were tested using multivariate logistic regression method to assess the predictive significance. Results: Out of 105 cases studied, 29 cases showed LN metastasis. Maximum numbers of cases affected were females with involvement of buccal mucosa. We found significant association of cervical LN metastasis with high grade of differentiation, lack of E-cadherin expression, high Ki-67 and cyclin D1 expression. In our study; tumor depth, thickness, extent of peritumoral lympho-plasmacytic infiltration, presence of eosinophils, tumor nest type, p53 and microvessel density (MVD) showed no significant correlation. Conclusion: Significant association of cervical LN metastasis with high grade of differentiation, lack of E-cadherin expression, high Ki-67 and cyclin D1 expression was seen.
Keywords: Cyclin D1, E-cadherin, grade of differentiation of OSCC, Ki-67, lymph node metastasis
|How to cite this article:|
Suresh T N, Hemalatha A, Harendra Kumar M L, Azeem Mohiyuddin S M. Evaluation of histomorphological and immunohistochemical parameters as biomarkers of cervical lymph node metastasis in squamous cell carcinoma of oral cavity: A retrospective study. J Oral Maxillofac Pathol 2015;19:18-24
|How to cite this URL:|
Suresh T N, Hemalatha A, Harendra Kumar M L, Azeem Mohiyuddin S M. Evaluation of histomorphological and immunohistochemical parameters as biomarkers of cervical lymph node metastasis in squamous cell carcinoma of oral cavity: A retrospective study. J Oral Maxillofac Pathol [serial online] 2015 [cited 2019 Mar 25];19:18-24. Available from: http://www.jomfp.in/text.asp?2015/19/1/18/157195
| Introduction|| |
Oral cancer is the sixth most common cancer worldwide with high prevalence in South Asia. , Oral cancers are most prevalent in Kolar and constitute 29.66% of total cancer incidence in Kolar.  Despite substantial development in both diagnosis and treatment in recent decades, the prognosis of the oral squamous cell carcinoma (OSCC) remains poor. Lymph node (LN) metastasis is shown to be the strongest prognostic indicator in OSCC. The 5- year survival drops considerably from 63 to 86% in patients with no nodal involvement to 20-36% in patients with LN metastasis. ,
Many methods are used to detect cervical LN metastasis. The sensitivity of preoperative imaging by computed tomography or magnetic resonance imaging and clinical examination is only around 70% and there is no imaging study capable of detecting micrometastasis in cervical LN.  Clinical assessment by the Tumor Node Metastasis (TNM) system is widely and routinely used to define the extent of tumor load and thus determines the treatment option for OSCC patients. Although 20-30% of clinically N0 patients will show occult metastasis on elective neck dissection, a considerably large number of patients will remain pathologically N0 and therefore do not benefit from neck dissection. , One of the major criticisms of the TNM system is that it ignores individual histological characteristic of tumor.  Therefore many studies proposed histological grading system to predict the biological behavior of LN metastasis. Multiple histological features have been tested over last 2 decades to predict LN metastasis that includes nuclear pleomorphism, mitotic index, lympho-plasmacytic response, pattern of invasion (POI), tumor nest, tumor thickness, tumor depth, lympho-vascular invasion, perineural invasion and grade of tumor. Many studies also used immunohistochemical (IHC) markers like p53, Ki-67, cyclinD1, epidermal growth factor receptor, CD31, cyclooxygenase 2, mucin 1(MUC1), laminin 5g2, E-cadherin and β-catenin to find out useful IHC marker to predict LN metastasis. ,,,, Aim of this study was to identify clinicopathological factors and IHC biomarkers in predicting cervical metastasis in OSCC patients.
| Materials and Methods|| |
Total number of 2,538 cases of malignancies were reported in the Department of Pathology, between January 2006 and December 2011. During study period, 523 patients were diagnosed as OSCC. A total of 115 patients underwent wide excision of primary tumor with simultaneous selective resection of regional LNs. In 105 cases, it was possible to retrieve surgical pathology material for review and to conduct IHC study. Patients with prior radiotherapy/chemotherapy/surgery for malignancy in head and neck region were excluded from the study. Clinically, TNM staging was done. Growth pattern of the tumor on gross examination was recorded. Tumors which were ulcerated, exophytic and cauliflower-like were described as ulceroproliferative. Ulcerated tumor which shows mainly an invasive component was termed as ulceroinfiltrative. Bulky exophytic tumor with superficial invasion and broad front were labeled as verrucous. Paraffin blocks and hematoxylin and eosin slides were retrieved from filing section of Department of Pathology. All the slides were reviewed by two pathologists without the knowledge of patient's clinical details and LN status.
Microscopic tumor thickness was defined as maximum tumor thickness excluding the keratin coat, taking the vertical extent of tumor from the surface to its deepest extent in a perpendicular fashion. Microscopic tumor depth was taken as infiltrative portion of the tumor which extended below the surface of the adjacent mucosa. Cases in which the epithelium was destroyed, it was measured after reconstituting a virtual surface. The starting points differed for these two measurements, but the deepest point of invasive tumor border was identical for both. In 18 cases, we could not measure the tumor depth, as adjacent normal mucosa was not identified on section.
Grade of differentiation was divided into well, moderate, or poor as described by Martinez-Gimeno et al.,  and the extent of peri tumoral lympho-plasmacytic infiltration was divided into three grades according to Brandwein-Gensler et al.  Grade1 is characterized by a continuous dense layer of lympho-plasmacytic infiltration among tumor and healthy tissue. Grade 2 shows a discontinuous patchy pattern of lympho-plasmacytic infiltration. Grade 3 shows only minimal or no lympho-plasmacytic infiltration.
POI types 1-4 are identified as defined by Bryne et al.  Type 1 represents tumor invasion in a broad pushing manner with a smooth outline. Type 2 represents tumor invasion with broad pushing fingers or separate large tumor islands with a stellate appearance. Type 3 represents invasive islands of tumor, >15cells/island. Type 4 represent tumor islands, <15 cells/island. 20% was used as the minimal cut off for incorporating into any particular type. The shape of tumor nest is classified as type A where tumor had oval shape or sheet like nest with a round margin (with >80% tumor area showing these features). Tumors that had asteroid shape tumor nest with a speculated margin or scattered small tumor nest (>20% tumor area showing these features) were classified as type B. 
IHC was performed using non-biotin polymer based HRP detection system. Slides were incubated with p53, Ki-67, CD31, cyclin D1 and E-cadherin primary antibodies (Biogenix, USA) at room temperature according to manufacturer's recommendation. Percentage of p53, Cyclin D1 and Ki-67 positive tumor cells were calculated by counting the number of brown-stained tumor nuclei in hotspots. In each case approximately 1000 tumor cells were examined. According to previous literature, we classified sample as positive if >10, >40 and >30% of tumor nuclei were stained by anti-p53, anti-cyclinD1 and Ki-67 antibody receptors, respectively. , Expression of E-cadherin was defined as high when membrane staining of >50% of cells was observed and low when membrane staining ≤50% of the cells stained. Small blood vessels were visualized by staining endothelial cells for CD31 antibodies. Microvessel density (MVD) was calculated as highest number of vessels on high power field (×40) at invasive front of tumor and the patients were then divided into two groups, with a median value as the dividing line. In calculating the MVD, areas of inflammation and adjacent benign tissues were excluded.
In order to explore the relation between clinical, histopathological parameters, IHC markers and the frequency of LN metastasis; chi-square test was used. In second step, all variables were tested in Multivariate logistic regression method to assess the predictive significance of above parameters. All calculations were carried out using statistical Package for Social Sciences (SPSS) version 16. Values of P < 0.05 were considered as statistically significant.
| Results|| |
A total of 105 patients (15 males and 90 females) with a mean age of 50.9 years (range 25-70 years) were included in this study. The site of primary origin was in buccal mucosa in 89 (84.7%) cases, lower alveolus in 12 (11.4%) cases, anterior two-third of the tongue in two cases and floor of mouth in two cases. Primary tumor was on the left side in 63 cases, on right side in 38 cases and in the midline in four cases. Clinical TNM staging showed six (5.7%) cases in Stage I, 22 (21.0%) cases in stage II, 31 (29.5%%) cases in stage III and 46 (43.8%) cases in stage IV. Out of 105 patients, 29 (27.61%) patients had cervical LN metastasis. On statistical analysis, sex and anatomical site showed no significant association with respect to node metastasis [Table 1].
|Table 1: Frequency distribution of clinical features with respect to node metastasis |
Click here to view
The distribution of histopathological parameters between tumor with and without cervical LN metastasis is summarized in [Table 2].
Grade of differentiation and POI showed significant correlation with the occurrence of cervical LN metastasis. Majority of well-differentiated SCC (78.8%) showed no LN metastasis as compared to high occurrence of metastasis in moderate-and poorly-differentiated SCC (55%). Highly infiltrating SCC (POI type 4) was significantly associated with higher likelihood of LN metastasis [Figure 1].
|Figure 1: Squamous cell carcinoma showing type 4 pattern of invasion (H&E stain, ×100). H&E = Hematoxylin and eosin|
Click here to view
The measurement of tumor thickness and tumor depth in primary tumor revealed an average value of 8.24 and 5.78 mm, respectively, with a range of 1-21 and 0-20 mm, respectively. However, none of the cut off values for tumor thickness or tumor depth achieved statistical significance to predict LN metastasis.
Lymphovascular invasion was found in two patients and both the patients showed LN metastasis. Perineural invasion associated with nodal metastasis was seen in three out of four patients. In our study extent of peritumoral lympho-plasmacytic infiltration, presence of eosinophils and tumor nest type showed no significant correlation.
A total of 904 LN were examined with an average of 8.6 per patient. Out of 282, 65 (23.0%) LNs studied in metastatic group showed metastasis.
Nuclear expression of p53, Ki-67 and cyclinD1 ranged from 0 to 95%, 5 to 70% and 5to 90%, respectively. E-cadherin expression ranged from 0 to 90%. MVD ranged from 2 to 56/HPF with a median of 12/HPF. Decreased expression of E-cadherin and increased Ki-67 and cyclin D1 expression was significantly associated with LN metastasis [Figure 2] [Figure 3] [Figure 4]. However, MVD and p53 expression showed no significant correlation [Table 3].
Multivariate analysis of clinico-pathological factors and IHC biomarkers showed association of cervical LN metastasis with high grade of differentiation, low E-cadherin expression, high Ki-67 and Cyclin D1 expression (P = 0.007, 0.001, 0.029 and 0.020, respectively).
|Figure 2: (a) Photomicrograph showing high expression of E-cadherin (IHC stain, x400). (b) Photomicrograph showing low expression of E-cadherin (IHC stain, x400)|
Click here to view
|Figure 3: Immunohistochemical staining of Ki-67 in squamous cell carcinoma showing increased mitotic activity (IHC stain, ×400)|
Click here to view
|Figure 4: Immunohistochemical staining of cyclin D1 in squamous cell carcinoma showing high expression (IHC stain, ×100)|
Click here to view
| Discussion|| |
Clinical TNM system is widely used by clinician for the management of OSCC patients. But it has been found that surface tumor size and size of the cervical LN does not correlate with the occurrence of metastasis in OSCC. , Studies have shown that LN in excess of 20mm may be histologically reactive hyperplasia without metastasis.  General policy of elective neck dissection based on clinical TNM staging exposes many OSCC patients to neck dissection that may not be necessary. Detailed pathological study of sentinel LN appears to be accurately predicting presence of metastasis and prevent the morbidity associated with unnecessary neck dissection in clinically N0/N1 patients.  However, sentinel LN biopsy in head and neck cancer is not the standard of care and is practiced only in few centers. The complex drainage pattern of head and neck region and proximity of LN to injection site raised concerns that sentinel LN may not be accurate in head and neck cancer. Many studies have investigated histopathological parameters, IHC and molecular markers for potential predictive factors. If these predictive factors accurately identify LN metastasis before neck dissection, the treatment would be more selective and overall cost and morbidity would be minimized.
We found significant correlation between grades of differentiation and cervical LN metastasis. Reports by Martinez-Gimeno et al., Byers et al., Sparano et al., Kurokawa et al. and Pimenta et al., also found a significant correlation between histological grades and occult nodal metastasis. ,,,, In the study by Chen et al., prevalence of nodal metastasis in elective neck dissection was 32% for well and 75% for poorly-differentiated carcinoma. 
In our study there was a significant correlation between POI and cervical LN metastasis. Only 20 out of 102 (19.6%) patients in low risk group (POI type 1, 2 and 3), but nine out of 13(69.2%) patients of high risk group (type 4) revealed cervical LN metastasis. Various other studies by Hiratsuka et al., Kurokawa et al., Osaki et al., Nagata et al., Okamoto et al., Goerkem et al. and Borges et al., showed POI/mode of invasion (MOI) as important predictor of cervical LN metastasis. ,,,,,,
Chang et al., proposed invasive pattern grading score (IPGS) which is based on Bryne et al.,'s criteria. , In their study, two most prevalent patterns at the invasive front of OSCC were considered similar to Gleason's score in carcinoma of prostate. Since each invasive score was assigned a number between 1 and 4,total summed score ranged from 2 to 8. Twenty percent was used as minimal cut off for incorporating into grading system. They found statistically significant correlation between IPGS, LN metastasis, distant metastasis and tumor recurrence. Highly significant association between presence of lymphovascular/perineurial invasion and LN metastasis was well established in the studies by Sparano et al., Pimenta et al., Borges et al., Shingaki et al., and Brown et al. ,,,,
In contradiction to the results published for elective neck dissection, neither tumor thickness nor tumor depth was significantly associated with cervical LN metastasis. Lim et al., Kurokawa et al. and Fakih et al., suggested tumor depth more than 4 mm, but Kane et al. and Fukano et al., found that tumor depth more than 5 mm carries a high risk for cervical LN metastasis. ,,,, In a study by Yuen et al., tumor thickness from 3 to 9 mm was associated with a 50% nodal metastasis rate; whereas, HoΊal et al ., found that thickness of ≥9 mm was the only variable that predicted occult metastas is in tongue carcinoma. , However, in Goerkem et al.,'s study of 78 patients no significant association between tumor thickness or tumor depth with cervical LN metastasis was reported.  Most of the above stated studies were done on carcinoma of tongue and floor of mouth. In our study, tongue carcinoma and carcinoma of floor of mouth were seen in only two cases each. Majority of our cases showed buccal carcinoma (83.8%). Carcinoma of buccal mucosa appears to behave differently in relation to metastatic potential as compared to carcinoma of tongue and floor of mouth. Tumor thickness and tumor depth which is widely described as predictor of cervical LN metastasis in tongue carcinoma may not be applicable to carcinoma of buccal mucosa. Hence, large prospective studies are required in buccal carcinoma to establish the significance of tumor thickness and tumor depth for predicting regional node involvement.
Oral carcinogenesis is a multistep process in which 6-10 genetic events leads to the disruption of the normal regulatory pathways that controls basic cellular functions including cell division, differentiation and cell death.  Mutation in the p53 tumor suppresser gene is the most common genetic change in human cancers and is regarded as an early event in carcinogenesis. , In our study, 99(94.2%) cases showed positivity to p53 antibodies. However, there was no significant difference between metastatic and non-metastatic group in our study.
E-cadherin is a calcium-dependent intracellular adhesion glycoprotein in epithelial cells. It plays a very important role in cell-cell adhesion, homotypic binding of epithelial tissue and also in its morphogenesis and cancer metastasis.  Several studies reported existence of association between low expression of E-cadherin expression and LN metastasis. ,, Wang et al., have found low expression of E-cadherin at the invasive front of the tumor than the central/superficial part in tumors with LN metastasis. It was also shown that low expression inversely correlated with invasive front grading system score, tumor thickness, poor survival rate and tumor size.  In our study we found a statistical significance association of low E-cadherin expression with LN metastasis (P = 0.001).
Ki-67 is a proliferation marker. High Ki-67 index is associated with poor prognosis in tongue SCC.  Study by Carlos et al., revealed a correlation between Ki-67 index and histological grades of differentiation in OSCC patients.  In our study, Ki-67 (>30% tumor cells) showed positivity in 49 (46.6%)cases. Ki-67 positivity statistically correlated with cervical LN metastasis (P = 0.005).
Cyclin D1 overexpression in carcinoma cells indicates accelerated G1 progression and entry into S phase of cell cycle with lower cell dependence on growth factors for proliferation. Cyclin D1 over expression has been reported in OSCC and is correlated with cytological grade, infiltrative pattern and metastasis. ,,, Carlos et al.,'s study showed that cyclin D1 expression was significantly associated with advanced tumor stage, LN metastasis and high Ki-67 index (>50% tumor positivity).  In our study, 47 (44.7%) cases showed cyclin D1 (>40% tumor cells) positivity and there was significantcorrelation with cervical LN metastasis (P = 0.0028).
In our study, MVD in the primary tumor did not show any significant correlation with metastasis. Few studies have reported that MVD acts as an independent prognostic factor and is also not associated with tumor grade. [53 ]
| Conclusion|| |
In literature there is a lacuna regarding the clinical and radiological factors that predict the cervical LN metastasis, hence there arises a need to evaluate histomorphological and immunohistochemical parameters as biomarkers of cervical LN metastasis. Our study shows that significant association of cervical LN metastasis exists with high grade of differentiation, lack of E-cadherin expression, high Ki-67 and cyclin D1 expression. An assessment of these factors in primary tumor may help us to predict LN metastasis, thereby minimizing the number of neck dissection.
| Acknowledgement|| |
We thank Sri Devaraj Urs Academy of Higher Education and Research Center (SDUAHER), Kolar for financial support (supply of Immunohistochemistry reagents) and authors sincerely thank Mr. Ravi Shankar S for helping us for statistical analysis.
| References|| |
Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 2009;45:309-16.
Moore MA, Ariyaratne Y, Badar F, Bhurgri Y, Datta K, Mathew A, et al
. Cancer epidemiology in South Asia-past, present and future. Asian Pac J Cancer Prev 2010;11:49-66.
Kalyani R, Das S, Bindra Singh MS, Kumar H. Cancer profile in the Department of Pathology of Sri Devaraj Urs Medical College, Kolar: A ten years study. Indian J Cancer 2010;47:160-5.
Kalnins IK, Leonard AG, Sako K, Razack MS, Shedd DP. Correlation between prognosis and degree of lymph node involvement in carcinoma of the oral cavity. Am J Surg 1977;134:450-4.
Grandi C, Alloisio M, Moglia D, Podrecca S, Sala L, Salvatori P, et al
. Prognostic significance of lymphatic spread in head and neck carcinomas: Therapeutic implications. Head Neck Surg 1985;8:67-73.
Woolgar JA. Pathology of the N0 neck. Br J Oral Maxillofac Surg 1999;37:205-9.
Byers RM, Wolf PF, Ballantyne AJ. Rationale for elective modified neck dissection. Head Neck Surg 1988;10:160-7.
van den Brekel MW, van der Waal I, Meijer CJ, Freeman JL, Castelijns JA, Snow GB. The incidence of micrometastases in neck dissection specimens obtained from elective neck dissections. Laryngoscope 1996;106:987-91.
Anneroth G, Batsakis J, Luna M. Review of the literature and a recommended system of malignancy grading in oral squamous cell carcinomas. Scand J Dent Res 1987;95:229-49.
Chang YC, Nieh S, Chen SF, Jao SW, Lin YL, Fu E. Invasive pattern grading score designed as an independent prognostic indicator in oral squamous cell carcinoma. Histopathology 2010;57:295-303.
Bryne M. Prognostic value of various molecular and cellular features in oral squamous cell carcinomas: A Review. J Oral Pathol Med 1991;20:413-20.
Odell EW, Jani P, Sherriff M, Ahluwalia SM, Hibbert J, Levison DA, et al
. The prognostic value of individual histologic grading parameters in small lingual squamous cell carcinomas. The importance of the pattern of invasion. Cancer 1994;74:789-94.
Schliephake H. Prognostic relevance of molecular markers of oral cancer-A review.Int J Oral Maxillofac Surg 2003;32:233-45.
Lim SC, Zhang S, Ishii G, Endoh Y, Kodama K, Miyamoto S, et al
. Predictive markers for late cervical metastasis in stage I and II invasive squamous cell carcinoma of the oral tongue. Clin Cancer Res 2004;10:166-72.
Martinez-Gimeno C, Rodriguez EM, Vila CN, Varela CL. Squamous cell carcinoma of the oral cavity: A clinicopathologic scoring system for evaluating risk of cervical lymph node metastasis. Laryngoscope 1995;105:728-33.
Brandwein-Gensler M, Teixeira MS, Lewis CM, Lee B, Rolnitzky L, Hille JJ,et al
. Oral squamous cell carcinoma: Histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival. Am J Surg Pathol 2005;29:167-78.
Bryne M, Koppang HS, Lilleng R, Kjaerheim A. Malignancy grading of the deep invasive margins of oral squamous cell carcinomas has high prognostic value. J Pathol 1992;166:375-81.
Nakanishi Y, Ochiai A, Kato H, Tachimori Y, Igaki H, Hirohashi S. Clinicopathological significance of tumor nest configuration in patients with esophageal squamous cell carcinoma. Cancer 2001;91:1114-20.
Yuen PW, Chow V, Choy J, Lam KY, Ho WK, Wei WI. The clinicopathologic significance of p53 and p21 expression in the surgical management of lingual squamous cell carcinoma. Am J ClinPathol 2001;116:240-5.
Hiratsuka H, Miyakawa A, Nakamori K,Sunakawa H, Kohama G. Multivariate analysis of occult lymph node metastasis as a prognostic indicator for patients with squamous cell carcinoma of the oral cavity. Cancer 1997;80:351-6.
Moore C, Kuhns JG,Greenberg RA. Thickness as prognostic aid in upper aerodigestive tract cancer. Arch Surg 1986;121:1410-4.
Friedman M, Roberts N, Kirshenbaum GL, Colombo J. Nodal size of metastatic squamous cell carcinoma of the neck. Laryngoscope 1993;103:854-6.
Suresh TN, Harendra Kumar ML, Thomas AK, Azeem M. Study of sentinel lymph node in oral Squamous cell carcinoma. J Clin Biomed Sci 2013;3:146-9.
Byers RM, El-Naggar AK, Lee YY, Rao B, Fornage B, Terry NH, et al
. Can we detect or predict the presence of occult nodal metastases in patients with squamous carcinoma of the oral tongue? Head Neck 1998;20:138-44.
Sparano A, Weinstein G, Chalian A, Yodul M, Weber R. Multivariate predictors of occult neck metastasis in early oral tongue cancer.Otolaryngol Head Neck Surg 2004;131:472-6.
Kurokawa H, Yamashita Y, Takeda S, Zhang M, Fukuyama H, Takahashi T. Risk factors for late cervical lymph node metastases in patients with stage I or II carcinoma of the tongue. Head Neck 2002;24:731-6.
Pimenta Amaral TM, Da Silva Freire AR, Carvalho AL, Pinto CA, Kowalski LP. Predictive factors of occult metastasis and prognosis of clinical stages I and II squamous cell carcinoma of the tongue and floor of the mouth. Oral Oncol 2004;40:780-6.
Chen RB, Suzuki K, Nomura T, Nakajima T. Flowcytometricanalysis of squamous cell carcinomas of the oral cavity in relation to lymph node metastasis. J Oral Maxillo fac Surg 1993;51:397-401.
Osaki T, Hirota J, Yoneda K, Yamamoto T, Ueta E.Clinical and histopathologic characteristics of tongue and gingiva carcinomas with occult and clinically evident cervical lymph-node metastasis. Int J Oral Maxillo Fac Surg 1996;25:274-8.
Nagata T, Schmelzeisen R, Mattern D, Schwarzer G, Ohishi M. Application of fuzzy inference to European patients to predict cervical lymph node metastasis in carcinoma of the tongue. Int J Oral Maxillo fac Surg 2005;34:138-42.
Okamoto M, Ozeki S, Watanabe T, Iida Y, Tashiro H. Cervical lymph node metastasis in carcinoma of the tongue. Correlation between clinical and histopathological findings and metastasis. J Craniomaxillo fac Surg 1988;16:31-4.
Goerkem M, Braun J, Stoeckli SJ. Evaluation of clinical and histomorphological parameters as potential predictors of occult metastases in sentinel lymph nodes of early squamous cell carcinoma of the oral cavity. Ann Surg Oncol 2010;17:527-35.
Borges AM, Shrikhande SS, Ganesh B. Surgical pathology of squamous carcinoma of the oral cavity: Its impact on management. Semin Surg Oncol 1989;5:310-7.
Shingaki S, Suzuki I, Nakajima T, Kawasaki T. Evaluation of histopathologic parameters in predicting cervical lymph node metastasis of oral and oropharyngeal carcinomas. Oral Surg Oral Med Oral Pathol 1988;66:683-8.
Brown B, Barnes L, Mazariegos J, Taylor F, Johnson J, Wagner RL. Prognostic factors in mobile tongue and floor of mouth carcinoma. Cancer 1989;64:1195-202.
Fakih AR, Rao RS, Borges AM, Patel AR. Elective versus therapeutic neck dissection in early carcinoma of the oral tongue. Am J Surg 1989;158:309-13.
Kane SV, Gupta M, Kakade AC, D' Cruz A. Depth of invasion is the most significant histological predictor of subclinical cervical lymph node metastasis in early squamous carcinomas of the oral cavity.Eur J Surg Oncol 2006;32:795-803.
Fukano H, Matsuura H, Hasegawa Y, Nakamura S. Depth of invasion as a predictive factor for cervical lymph node metastasis in tongue carcinoma. Head Neck 1997;19:205-10.
Yuen AP, Lam KY, Wei WI, Lam KY, Ho CM, Chow TL, et al
. A comparison of the prognostic significance of tumor diameter, length, width, thickness, area, volume and clinicopathological features of oral tongue carcinoma. Am J Surg 2000;180:139-43.
Hoºal AS, Unal OF, Ayhan A. Possible prognostic value of histopathologic parameters in patients with carcinoma of the oral tongue. Eur Arch Otorhinolaryngol 1998;255:216-9.
Williams HK. Molecular pathogenesis of oral squamous carcinoma. Mol Pathol 2000;53:165-72.
Kannan S, Chandran GJ, Pillai KR, Mathew B, Sujathan K, Nalina kumary KR, et al
. Expression of p53 in leukoplakia and squamous cell carcinoma of the oral mucosa: Correlation with expression of Ki67. Clin Mol Pathol 1996;49:M170-5.
Levine AJ, Perry ME, Chang A, Silver A, Dittmer D, Wu M, et al
. The 1993 Walter Hubert Lecture: The role of the p53 tumour-suppressor gene in tumorigenesis. Br J Cancer 1994;69:409-16.
Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991;251:1451-5.
Lopes FF, da Costa Miguel MC, Pereira AL, da Cruz MC, de Almeida Freitas R, Pinto LP, et al
. Changes in immune expression of E-cadherin and beta-catenin in oral squamous cell carcinoma with and without nodal metastasis. Ann Diagn Pathol 2009;13:22-9.
Chow V, Yuen AP, Lam KY, Tsao GS, Ho WK, Wei WI. A comparative study of the clinicopathological significance of E-cadherin and catenins (alpha, beta, gamma) expression in the surgical management of oral tongue carcinoma. J Cancer Res Clin Oncol 2001;127:59-63.
Wang X, Zhang J, Fan M, ZhouQ, Deng H, Aisharif MJ, et al
. The expression of E-cadherin at the invasive tumor front of oral squamous cell carcinoma: Immunohistochemical and RTPCR analysis with clinicopathological correlation.Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:547-54.
Xie X, De Angelis P, Clausen OP, Boysen M. Prognostic significance of proliferative and apoptotic markers in oral tongue squamous cell carcinomas. Oral Oncol 1999;35:502-9.
Carlos de Vicente J, Herrero-Zapatero A, Fresno MF, López-Arranz JS. Expression of cyclin D1 and Ki-67 in squamous cell carcinoma of the oral cavity: Clinicopathological and prognostic significance. Oral Oncol 2002;38:301-8.
Salesiotis AN, Cullen KJ. Molecular markers predictive of response and prognosis in the patient with advanced squamous cell carcinoma of the head and neck: Evolution of a model beyond TNM staging. Curr Opin Oncol 2000;12:229-39.
Kuo MY, Lin CY, Hahn LJ, Cheng SJ, Chiang CP. Expression of cyclin D1 is correlated with poor prognosis in patients with areca quid chewing-related oral squamous cell carcinomas in Taiwan. J Oral Pathol Med 1999;28:165-9.
Mineta H, Miura K, Takebayashi S, Ueda Y, Misawa K, Harada H, et al
. Cyclin D1 overexpression correlates with poor prognosis in patients with tongue squamous cell carcinoma. Oral Oncol 2000;36:194-8.
Kukreja I, Kapoor P, Deshmukh R, Kulkarni V. VEGF and CD 34: A correlation between tumor angiogenesis and microvessel density - An immunohistochemical study. J Oral Maxillofac Pathol 2013;17:367-73.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]