“a study of expression of e cadherin and...

“A STUDY OF EXPRESSION OF E CADHERIN AND

VIMENTIN IN ORAL SQUAMOUS CELL CARCINOMA”

Dissertation submitted in

partial fulfilment of the requirements for the degree of

M.D. (PATHOLOGY)

BRANCH – III

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

CHENNAI

KARPAGA VINAYAGA INSTITUTE OF MEDICAL

SCIENCES AND RESEARCH CENTRE

MADURANTHAGAM - 603308

APRIL’ 2017

CERTIFICATE

This is to certify that this Dissertation entitled “A STUDY OF

EXPRESSION OF E CADHERIN AND VIMENTIN IN ORAL

SQUAMOUS CELL CARCINOMA” is the bonafide original work of

Dr.INDUMATI .B., in partial fulfillment of the requirement for M.D., (Branch

III) in Pathology examination of the Tamilnadu Dr.M.G.R Medical University

to be held in April 2017.

Prof. Dr.CHITRA .T, M.D., Prof. Dr.A.B.HARKE, M.D.,

PROFESSOR AND HOD PROFESSOR OF PATHOLOGY,

DEPARTMENT OF PATHOLOGY KARPAGA VINAYAGA INSTITUTE OF

KARPAGA VINAYAGA INSTITUTE OF MEDICAL SCIENCES AND

MEDICAL SCIENCES AND RESEARCH CENTRE

RESEARCH CENTRE MADURANTHAGAM – 603 308.

MADURANTHAGAM – 603 308.

Prof. Dr.SUFULA SUNIL VISWAS RAO, M.D.,

PRINCIPAL,

KARPAGA VINAYAGA INSTITUTE OF

MEDICAL SCIENCES AND RESEARCH CENTRE

MADURANTHAGAM – 603308.

DECLARATION

I, Dr. Indumati.B, solemnly declare that the dissertation titled

“A STUDY OF EXPRESSION OF E CADHERIN AND VIMENTIN IN

ORAL SQUAMOUS CELL CARCINOMA” is the bonafide work done by

me at Department of Pathology, Karpaga vinayaga Institute of Medical

Sciences and Research Centre, under the expert guidance and supervision of

Prof. Dr. Chitra .T, M.D., Professor and HOD of Pathology, Karpaga

Vinayaga Medical College. The dissertation is submitted to the Tamilnadu

Dr. M.G.R Medical University towards partial fulfillment of requirement for

the award of M.D., Degree (Branch III) in Pathology.

Place: Maduranthagam

Date: Dr. INDUMATI.B

ACKNOWLEDGEMENT

I express my sincere thanks to Dr.R.ANNAMALAI Managing Director

of Karpaga Vinayaga Institute of Medical Sciences and Research, for

permitting me to utilize the facilities of the Institution.

I thank Prof. Dr.SUFULA SUNIL VISWAS RAO M.D., Principal,

Karpaga Vinayaga Institute of Medical Sciences and Research Centre for

helping me to carryout the dissertation. I take the opportunity to express my

heartiest thanks to Prof. Dr. CHITRA.T, M.D., Professor and HOD,

Department of Pathology, Karpaga Vinayaga Medical College, for her keen

interest, constant encouragement and valuable suggestions throughout the

study.

I am extremely thankful to Dr. A.B.HARKE, M.D., Professor of

Pathology, Karpaga Vinayaga Medical College, for his valuable suggestions,

constant support, advice and encouragements throughout the study.

I am truly thankful to all faculties in department of OMFS in Karpaga

Vinayaga Dental College.

I express my heartfelt sincere thanks to all my Assistant Professors for

their help and suggestions during the study.

I am thankful to my colleagues, friends, technicians and staff of the

department of Pathology, for all their help and support they extended for the

successful completion of this dissertation.

I thank Dr.SATHYANARAYANAN of Karpaga Vinayaga Institute for

rendering me great support to finish the dissertation.

I express my heartfelt thanks to Dr.ARUN for helping me out in

statistics. I am so honoured to consider my family close to my heart for

rendering all the support for completing this successfully.

ABBREVIATIONS

SCC : Squamous cell carcinoma

HNSCC : Head and Neck squamous cell carcinoma

EGFR : Epidermal growth factor receptor

WHO : World Health Organisation

TGFβ : Transforming growth factor

PCNA : Proliferating Cell Nuclear Antigen

AJCC : American Joint Committee on Cancer

IHC : Immunohistochemistry

LI : Labeling Index

H & E : Hematoxylin & Eosin

HPV : Human papilloma virus

SIN : Squamous intraepithelial neoplasia

CIS : Carcinoma in situ

CYP1A1 : Cytochrome P450 1A1

GSTM1 : Glutathione S-transferase mu 1

OSCC : Oral squamous cell carcinoma

PCR : Polymerase chain reaction

N : Number of Cases

CONTENTS

S.NO. TITLE PAGE NO.

1 INTRODUCTION 1

2 AIMS AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4

4 MATERIALS AND METHODS 35

5 OBSERVATION AND RESULTS 40

6 DISCUSSION 69

7 SUMMARY 75

8 CONCLUSION 77

BIBLIOGRAPHY

ANNEXURES

MASTER CHART

BIBLIOGRAPHY

1. Warnakulasuriya. S: Global epidemiology of oral and oropharyngeal

cancer. Oral Oncol 2009; 45 (4–5): 309–16.

2. Anastasios K. Markopoulos. Current Aspects on Oral Squamous Cell

Carcinoma. The Open Dentistry Journal. 2012; 6: 126-130.

3. Cooper JS, Porter K, Mallin K, Hoffman HT, Weber RS, Ang KK, Gay

EG, Langer CJ, National Cancer Database report on cancer of the head

and neck: 10-year update, Head Neck, 2009; 31(6):748– 758.

4. Barnes L, Eveson JW, Reichart P, Sidransky D (eds), Pathology and

genetics of head and neck tumours, World Health Organization

Classification of Tumors, IARC Press; Lyon, 2005.

5. Banerjee AG, Bhattacharyya I, Vishwanatha JK. Identification of genes

and molecular pathways involved in the progression of premalignant

oral epithelia. Mol Cancer Ther 2005; 4: 865-75.

6. Paul M. Speight. Update on Oral Epithelial Dysplasia and Progression

to Cancer. Head and Neck Pathol (2007) 1:61–66.

7. Bouquot J, Speight PM, Farthing PM. Epithelial dysplasia of the oral

mucosa - diagnostic problems and prognostic features. Curr Diagn

Pathol 2006; 12:11–22.

8. S. Humayun and V. Ram Prasad. Expression of p53 protein and ki-67

antigen in oral premalignant lesions and oral squamous cell carcinomas:

An immunohistochemical study. Natl J Maxillofac Surg. 2011

Jan-Jun; 2(1): 38–46.

9. Bina Raju, Ravi Mehrotra, Gunnvor Oijordsbakken, Ali K.Al- Sharabi,

Endre N. Vasstrand and Salah O.Ibrahim. Expression of p53, Cyclin D1

and Ki-67 in Pre-malignant and Malignant Oral Lesions: Association

with Clinicopathological Parameters. Anticancer Research 2005;

25: 4699-4706.

10. Noushin Jalayer Naderi, Farrokh Tirgari, Farzin Esmaili, Faranak

Paktinat, and Zahra Keshavarz. Vascular Endothelial Growth Factor and

Ki-67 Antigen Expression in Relation to Age and Gender in Oral

Squamous Cell Carcinoma. J Dent Res Dent Clin Dent Prospect. 2012

summer; 6(3): 103–107.

11. Nanci A. Ten Cate’s Oral Histology: Development, Structure and

Function, 7th edn. St Louis, MO: Mosby Elsevier, 2008: 319-357.

12. Thomson PJ, Potten CS, Appleton DR. In vitro labeling studies and the

measurement of epithelial cell proliferative activity in the human oral

cavity. Arch Oral Biol 2001; 46: 1157-1164.

13. Petti S. Pooled estimate of world leukoplakia prevalence: systematic

review. Oral Oncol 2003; 39: 770-780.

14. Schepman KP, Bezemer PD, van der Meji EH, Smeele LE, vander Waal

I. Tobacco usage in relation to the anatomical site of oral leukoplakia.

Oral Dis 2001; 7: 25-27.

15. Liu SC, Klein-Szanto AJ. Markers of proliferation in normal and

leukoplakic oral epithelia. Oral Oncol. 2000; 36:145–51.

16. Mehta FS, Shroff BC, Gupta PC,Daftary DK. Oral leukoplakia in

relation to tobacco haboits. A ten year follow up study of Bombay

policemen. Oral Surg Oral Med Pathol 1972; 34: 426-33.

17. Silverman S, Bhargava K, Smith LW, Malaowala AM. Malignant

transformation and natural history of oral leukoplakia in 57518

industrial workers of Gujarat, India. Cancer 1976; 38:1790-4.

18. Pindborg JJ, Reichart PA, Smith CJ, van der Waal I. Histological typing

of cancer and precancer of the oral mucosa. International histological

classification of tumours. 2nd ed. Springer: world health

organization; 1997.

19. Wain SL, Kier R, Vollmer RT, Bossen EH. Basaloid-squamous

carcinoma of the tongue, hypopharynx, and larynx: report of 10 cases.

Hum Pathol 1986; 17(11):1158–66.

20. Banks ER, Frierson HF Jr, Mills SE, George E, Zarbo RJ, Swanson PE.

Basaloid squamous cell carcinoma of the head and neck. A

clinicopathologic and immunohistochemical study of 40 cases. Am J

Surg Pathol 1992; 16(10):939–46.

21. Gonzalez-Moles MA, Esteban F, Rodriguez-Archilla A, Ruiz- Avila I,

Gonzalez-Moles S: Importance of tumour thickness measurement in

prognosis of tongue cancer. Oral Oncol 2002;38(4):394–7.

22. Woolgar JA. Histopathological prognosticators in oral and

oropharyngeal squamous cell carcinoma. Oral Oncol 2006;42(3):

229–39.

23. Jakobsson PA, Eneroth CM, Killander D, Moberger G, Mårtensson B.

Histologic classification and grading of malignancy in carcinoma of the

larynx (a pilot study) Acta Radiol Ther Phy Biol. 1973;12:1–8.

24. Anneroth G, Hansen LS. A methodologic study of histologic

classification and grading of malignancy in oral squamous cell

carcinoma. Scand J Dent Res. 1984; 92: 448–68.

25. Roland NJ, Caslin AW, Nash J, Stell PM. Value of grading squamous

cell carcinoma of the head and neck.Head Neck.1992;14: 224–9.

26. M Akhter, S Hossain, Quazi B Rahman, and Motiur R Molla A study on

histological grading of oral squamous cell carcinoma and its co-

relationship with regional metastasis J Oral Maxillofac Pathol. 2011;

15(2): 168–176. 27. Bryne M., Stromme H., Lilleng R., Stene T., Bang

G., Dabelsteen E. Bryne M. New malignancy grading is a better

prognostic indicator than Broder’s grading in oral squamous cell

carcinoma. J Oral Pathol Med 1989; 18: 432-7.

27. Frierson Jr HF, Cooper PH: Prognostic factors in squamous cell

carcinoma of the lower lip. Hum Pathol 1986; 17:346-354.

28. Iro H, Waldfahrer F: Evaluation of the newly updated TNM

classification of head and neck carcinoma with data from 3247 patients.

Cancer 1998; 83:2201-2207.

29. 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- 381

30. Dorta RG, Landman G, Kowalski LP, Lauris JRP, Latorre MRDO,

Oliveira DT: Tumor-associated tissue eosinophilia as a prognostic factor

in oral squamous cell carcinomas. Histopathology 2002; 41:152-157.

31. Kato H, Shi W, Hui A, PerezOrdonez B, Huang S, OSullivan B, W

aldron J, Gullane P, Gilbert R, Liu F-F: P16 over-expression is a

favorable prognostic factor in squamous cell carcinoma of the

oropharynx. Lab Invest 2009; 89:249A.

32. Miller K, Auld J, Jessup E, Rhodes A, Antigen unmarking by pressure

cooker method. A comparison with microwave oven heating and

traditional methods, Advances of anatomical pathology, 2:60-64.

References from 2nd

article

33. Weigelt B, Peterse JL, Van‘t Veer LJ. Breast cancer metastasis: markers

and models. Nat Rev Cancer 2005; 5: 591–602.

34. Citron ML, Berry DA, Cirrincione C et al. Randomized trial of dose-

dense versus conventionally scheduled and sequential versus concurrent

combination chemotherapy as postoperative adjuvant treatment of node-

positive primary breast cancer: first report of Intergroup Trial

C9741/Cancer and Leukemia Group B Trial 9741. J Clin Oncol 2003;

21: 1431–9.

35. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth

of cancer cells in metastatic sites. Nat Rev Cancer 2002; 2: 563–72.

36. Woodhouse EC, Chuaqui RF, Liotta LA. General mechanisms of

metastasis. Cancer 1997; 80: 1529–37.

37. Mehes G, Witt A, Kubista E, Ambros PF. Circulating breast cancer cells

are frequently apoptotic. Am J Pathol 2001; 159: 17–20.

38. Polyak K, Weinberg RA. Transitions between epithelial and

mesenchymal states: acquisition of malignant and stem cell traits. Nat

Rev Cancer 2009; 9: 265–73.

39. Thiery JP. Epithelial-mesenchymal transitions in tumour progression.

Nat Rev Cancer 2002; 2: 442–54.

40. Ngan CY, Yamamoto H, Seshimo I et al. Quantitative evaluation of

vimentin expression in tumour stroma of colorectal cancer. Br J Cancer

2007; 96: 986–92.

41. Raymond WA, Leong AS. Vimentin – a new prognostic parameter in

breast carcinoma? J Pathol 1989; 158: 107–14.

42. Dorudi S, Sheffield JP, Poulsom R, Northover JM, Hart IR. E-cadherin

expression in colorectal cancer. An immunocytochemical and in situ

hybridization study. Am J Pathol 1993; 142: 981–6.

43. Kowalski PJ, Rubin MA, Kleer CG. E-cadherin expression in primary

carcinomas of the breast and its distant metastases. Breast Cancer Res

2003; 5: R217–22.

44. Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour

progression: an alliance against the epithelial phenotype? Nat Rev

Cancer 2007; 7: 415–28.

45. Gregory PA, Bert AG, Paterson EL et al. The miR-200 family and miR-

205 regulate epithelial to mesenchymal transition by targeting ZEB1

and SIP1. Nat Cell Biol 2008; 10: 593–601.

46. Korpal M, Lee ES, Hu G, Kang Y. The miR-200 family inhibits

epithelial-mesenchymal transition and cancer cell migration by direct

targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J

Biol Chem 2008; 283: 14910–4.

47. Park SM, Gaur AB, Lengyel E, Peter ME. The miR-200 family

determines the epithelial phenotype of cancer cells by targeting the E-

cadherin repressors ZEB1 and ZEB2. Genes Dev 2008; 22: 894–907.

48. Mani SA, Guo W, Liao MJ et al. The epithelial-mesenchymal transition

generates cells with properties of stem cells. Cell 2008; 133: 704–15.

49. Greenburg G, Hay ED. Epithelia suspended in collagen gels can lose

polarity and express characteristics of migrating mesenchymal cells. J

Cell Biol 1982; 95: 333–9.

50. Hay ED. The mesenchymal cell, its role in the embryo, and the

remarkable signaling mechanisms that create it. Dev Dyn 2005;

233: 706–20.

51. Lai-kui Liu,Xiao-Yun Jiang et al ,Upregulation of vimentin and aberrant

expression of E cadherin in oral squamous cell carcinomas:correlation

with the clinicopathological features and patient outcome,modern

pathology(2010)23,213-224;doi:10.1038/modpathol.2009.160

52. Voulgari A, Pintzas A. Epithelial-mesenchymal transition in cancer

metastasis: mechanisms, markers and strategies to overcome drug

resistance in the clinic. Biochim Biophys Acta 2009; 1796: 75–90.

53. Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal

transitions. Oncogene 2005; 24: 5764–74.

54. Moustakas A, Heldin CH. Signaling networks guiding epithelial-

mesenchymal transitions during embryogenesis and cancer progression.

Cancer Sci 2007; 98: 1512–20.

55. Boyer B, Valles AM, Edme N. Induction and regulation of epithelial-

mesenchymal transitions. Biochem Pharmacol 2000; 60: 1091–9.

56. Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the

crossroads of development and tumor metastasis. Dev Cell 2008; 14:

818–29.

57. Chan AO, Chu KM, Lam SK et al. Soluble E-cadherin is an

independent pretherapeutic factor for long-term survival in gastric

cancer. J Clin Oncol 2003; 21: 2288–93.

58. Gould Rothberg BE, Bracken MB. E-cadherin immunohistochemical

expression as a prognostic factor in infiltrating ductal carcinoma of the

breast: a systematic review and meta-analysis. Breast Cancer Res Treat

2006; 100: 139–48.

59. Becker KF, Atkinson MJ, Reich U et al. E-cadherin gene mutations

provide clues to diffuse type gastric carcinomas. Cancer Res 1994;

54: 3845–52.

60. Berx G, Cleton-Jansen AM, Nollet F et al. E-cadherin is a

tumour/invasion suppressor gene mutated in human lobular breast

cancers. EMBO J 1995; 14: 6107–15.

61. Graff JR, Herman JG, Lapidus RG et al. E-cadherin expression is

silenced by DNA hypermethylation in human breast and prostate

carcinomas. Cancer Res 1995; 55: 5195–9.

62. Yoshiura K, Kanai Y, Ochiai A, Shimoyama Y, Sugimura T, Hirohashi

S. Silencing of the E-cadherin invasion-suppressor gene by CpG

methylation in human carcinomas. Proc Natl Acad Sci U S A 1995;

92: 7416–9.

63. Miettinen PJ, Ebner R, Lopez AR, Derynck R. TGF-beta induced

transdifferentiation of mammary epithelial cells to mesenchymal cells:

involvement of type I receptors. J Cell Biol 1994; 127: 2021–36.

64. Radisky DC, Kenny PA, Bissell MJ. Fibrosis and cancer: do

myofibroblasts come also from epithelial cells via EMT? J Cell

Biochem 2007; 101: 830–9.

65. Lewis MP, Lygoe KA, Nystrom ML et al. Tumour-derived TGF-beta1

modulates myofibroblast differentiation and promotes HGF/SF-

dependent invasion of squamous carcinoma cells. Br J Cancer 2004;

90: 822–32.

66. Wahid A, Ahamd S, Sajjad M. Pattern of carcinoma of oral cavity

reporting at dental department of Ayub medical college. J Ayub Med

Coll Abbottabad. 2005:17(1):65-6

67. Jung JW, Hwang SY, Hwang JS, Oh ES, Park S, Han IO. Ionising

radiation induces changes associated with epithelial-mesenchymal

transdifferentiation and increased cell motility of A549 lung epithelial

cells. Eur J Cancer 2007; 43: 1214–24.

68. Yang MH, Wu MZ, Chiou SH et al. Direct regulation of TWIST by

HIF-1alpha promotes metastasis. Nat Cell Biol 2008; 10: 295–305.

69. Wang X, Ling MT, Guan XY et al. Identification of a novel function of

TWIST, a bHLH protein, in the development of acquired taxol

resistance in human cancer cells. Oncogene 2004; 23: 474–82.

70. Yang AD, Fan F, Camp ER et al. Chronic oxaliplatin resistance induces

epithelial-to-mesenchymal transition in colorectal cancer cell lines. Clin

Cancer Res 2006; 12: 4147–53.

71. Kajiyama H, Shibata K, Terauchi M et al. Chemoresistance to paclitaxel

induces epithelial-mesenchymal transition and enhances metastatic

potential for epithelial ovarian carcinoma cells. Int J Oncol 2007;

31: 277–83.

72. Shah AN, Summy JM, Zhang J, Park SI, Parikh NU, Gallick GE.

Development and characterization of gemcitabine-resistant pancreatic

tumor cells. Ann Surg Oncol 2007; 14: 3629–37.

73. Hurwitz H, Fehrenbacher L, Novotny W et al. Bevacizumab plus

irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer.

N Engl J Med 2004; 350: 2335–42.

74. Piccart-Gebhart MJ, Procter M, Leyland-Jones B et al. Trastuzumab

after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J

Med 2005; 353: 1659–72.

75. Ishii H, Iwatsuki M, Ieta K et al. Cancer stem cells and chemoradiation

resistance. Cancer Sci 2008; 99: 1871–7.

76. Christiansen JJ, Rajasekaran AK. Reassessing epithelial to

mesenchymal transition as a prerequisite for carcinoma invasion and

metastasis. Cancer Res 2006; 66: 8319–26.

77. Garber K. Epithelial-to-mesenchymal transition is important to

metastasis, but questions remain. J Natl Cancer Inst 2008; 100:

232–3. 9.

78. Masaki T, Goto A, Sugiyama M et al. Possible contribution of CD44

variant 6 and nuclear beta-catenin expression to the formation of

budding tumor cells in patients with T1 colorectal carcinoma. Cancer

2001; 92: 2539–46.

79. AbstractFull Article (HTML)PDF(487K) References Web of Science®

Times Cited: 23

80. Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T. Opinion:

migrating cancer stem cells – an integrated concept of malignant tumour

progression. Nat Rev Cancer 2005; 5: 744–9.

81. Forastiere AA, Ang K, Brizel D, et al. Head and neck cancers. J Natl

Compr Canc Netw 2005;3:316–391.

82. Hunter KD, Parkinson EK, Harrison PR. Profiling early head and neck

cancer. Nat Rev Cancer 2005;5:127–135.

83. Casiglia J, Woo SB. A comprehensive review of oral cancer. Gen Dent

2001; 49:72–82.

84. Vokes EE, Weichselbaum RR, Lippman SM, et al. Head and neck

cancer. N Engl J Med 1993;328:184–194.

85. Bettendorf O, Piffkò J, Bànkfalvi A. Prognostic and predictive factors in

oral squamous cell cancer: important tools for planning individual

therapy? Oral Oncol 2004;40:110–119.

86. Bryne M, Koppang HS, Lilleng R, et al. Malignancy grading of the deep

invasive margins of oral squamous cell carcinomas has high prognostic

value. J Pathol 1992;166:375–381.

87. Woolgar JA. Histopathological prognosticators in oral and

oropharyngeal squamous cell carcinoma. Oral Oncol 2006;42:229–239.

88. Mandal M, Myers JN, Lippman SM, et al. Epithelial to mesenchymal

transition in head and neck squamous carcinoma: association of Src

activation with E-cadherin down-regulation, vimentin expression, and

aggressive tumor features. Cancer 2008;112:2088–2100.

89. Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-

mesenchymal transition during tumor progression. Curr Opin Cell Biol

2005;17:548–558.

90. Christiansen JJ, Rajasekaran AK. Reassessing epithelial to

mesenchymal transition as a prerequisite for carcinoma invasion and

metastasis. Cancer Res 2006;66:8319–8326.

91. Thiery JP. Epithelial-mesenchymal transitions in development and

pathologies. Curr Opin Cell Biol 2003;15:740–746.

92. Nawshad A, LaGamba D, Polad A, et al. Transforming growth factor-β

Signaling during epithelial-mesenchymal transformation: implications

for embryogenesis and tumor metastasis. Cells Tissues Organs

2005;179:11–23.

93. Behrens J. Cadherins and catenins: role in signal transduction and tumor

progression. Cancer Metastasis Rev 1999;18:15–30.

94. Hsu YM, Chen YF, Chou ChY, et al. KCl cotransporter-3 down-

regulates E-cadherin/β-catenin complex to promote epithelial-

mesenchymal transition. Cancer Res 2007;67:11064–11073.

95. Zheng Z, Pan J, Chu B, et al. Downregulation and abnormal expression

of E-cadherin and β-catenin in nasopharyngeal carcinoma: close

association with advanced disease stage and lymph node metastasis.

Hum Pathol 1999;30:458–466.

96. Pyo SW, Hashimoto M, Kim YS, et al. Expression of E-cadherin, P-

cadherin and N-cadherin in oral squamous cell carcinoma: correlation

with the clinicopathologic features and patient outcome. J

Craniomaxillofac Surg 2007;35:1–9.

97. Chang HW, Chow V, Lam KY, et al. Loss of E-cadherin expression

resulting from promoter hypermethylation in oral tongue carcinoma and

its prognostic significance. Cancer 2002; 94:386–392.

98. Diniz-Freitas M, García-Caballero T, Antúnez-López J, et al. Reduced

E-cadherin expression is an indicator of unfavourable prognosis in oral

squamous cell carcinoma. Oral Oncol 2006; 42:190–200.

99. Mahomed F, Altini M, Meer S. Altered E-cadherin/β-catenin expression

in oral squamous carcinoma with and without nodal metastasis. Oral Dis

2007; 13:386–392.

100. Tanaka N, Odajima T, Ogi K, et al. Expression of E-caderin, α-

catenine, and β-cetenin in the process of lymph node metastasis in oral

squamous cell carcinoma. Br J Cancer 2003; 89:557–563.

101. Andrews NA, Jones AS, Helliwell TR, et al. Expression of the E-

cadherin-catenin cell adhesion complex in primary squamous cell

carcinomas of the head and neck and their nodal metastases. Br J Cancer

1997; 75:1474–1480.

102. Chow V, Yuen AP, Lam KY, et al. A comparative study of the

clinicopathological significance of E-cadherin and catenins (α,β,γ)

expression in the surgical management of oral tongue carcinomas.

J Cancer Res Clin Oncol 2001;127:59–63.

103. Bánkfalvi A, Krassort M, Végh A, et al. Deranged expression of the

E-cadherin/beta-catenin complex and the epidermal growth factor

receptor in the clinical evolution and progression of oral squamous cell

carcinomas. J Oral Pathol Med 2002;31:450–457.

104. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic

regulator. Science 1991;251:1451–1455.

105. Behrens J, von Kries JP, Kühl M, et al. Functional interaction of β-

catenin with the transcription factor LEF-1. Nature 1996;382:638–642.

106. Conacci-Sorrell M, Zhurinsky J, Ben Ze'ev A. The cadherin-catenin

adhesion system in signaling and cancer. J Clin Invest 2002;109:

987–991.

107. Steinert PM, Roop DR. Molecular and cellular biology of intermediate

filaments. Annu Rev Biochem 1988;57:593–625.

108. Ramaekers FC, Haag D, Kant A, et al. Coexpression of keratin- and

vimentin-type intermediate filaments in human metastatic carcinoma

cells. Proc Natl Acad Sci USA 1983;80:2618–2622.

109. Gilles C, Polette M, Piette J, et al. Vimentin expression in cervical

carcinomas: association with invasive and migratory potential. J Pathol

1996;180:175–180.

110. Gilles C, Polette M, Zahm JM, et al. Vimentin contributes to human

mammary epithelial cell migration. J Cell Sci 1999;112:4615–4625.

111. Paccione RJ, Miyazaki H, Patel V, et al. Keratin down-regulation in

vimentin-positive cancer cells is reversible by vimentin RNA

interference, which inhibits growth and motility. Mol Cancer Ther

2008;7:2894–2903.

112. Gilles C, Polette M, Mestdagt Me, et al. Transactivation of vimentin

by β-catenin in human breast cancer cells. Cancer Res 2003;63:

2658–2664.

113. Bryne M, Boysen M, Alfsen CG, et al. The invasive front of

carcinomas. The most important area for tumor prognosis? Anticancer

Res 1998; 18:4757–4764.

114. Bànkfalvi A, Piffkò J. Prognostic and predictive factors in oral cancer:

the role of the invasive tumor front. J Oral Pathol Med 2000;

29:291–298.

115. Konrad steinestel, Stefan eder et al clinical significance of epithelial

mesenchymal transition.springer open journal 2014,3:17.

116. Lamouille s,xu j, Derynck r: molecular mechanism of epithelial

mesenchymal transition .nt rev mol cell boil 2014,15:178-196.

117. Hazan R B, Qiao R , Keren R, badano I, et al cadherin switch in tumor

progression ann n y acad sci 2004,1014:155-163.

118. Bourboulla d,settler Stevenson et al matrix mettaloproteinases in

tumor cell adhesion in seminars in cancer biology, Amsterdam;

Elsevier; 2010:161-168.

119. Yoo ya, Kang mh et al sonuc hedgehog pathway in metastasis and

lymphangiogenesis via emt cancer res 2011,71:7061-7070.

120. V.C.DE araujo, D.S.Pinto et al vimentin I n oral squamous cell

carcinoma’european archives otorhinolaryngology 1993,250:105-109.

121. Maria Carmen, Antonio et al ihc expression of e cadherin and cd44v6

in scc braz dent j.vol20, 2009, no.1 lai kui liu,xiao et al upregulation of

vimentin and aberrant expression of e cadherin modern pathology

2010,23,213-224

122. Jing ping, Detao et al expression of e cad and vim in oral scc ,ijclin

exp path 2015:8(3):3150-3154

123. Gurkiran kaur, Sumitha et al expre of e cad in primary prla scc ij of

dent res 2009,vol,20;1,71-76.

124. Shubha P Bhat, Ramesh Naik C N, G K Swetadri, Hilda D’souza,

Jayaprakash C S, Vadisha Bhat. Clinicopathological Spectrum of

Malignancies of Oral Cavity and Oropharynx- Our Experience in a

Referral Hospital.World articles in ear,nose & throat archives

2010: Vol 3.

125. Patel MM and Pandya AN. Relationship of oral cancer with age, sex,

site distribution and habits. Indian J Pathol Microbiol 2004; 47(2):

195-197.

126. Mehrotra R, Singh M, Kumar D, Pandey AN, Gupta RK, Sinha US.

Age specific incidence rate and pathological spectrum of oral cancer in

Allahabad. Indian J Med Sci. 2003 Sep; 57(9):400-4.

127. Jianming zhang ,Wei zhang ,Ping gao et al –expression of e cadherin

in oral squamous cell carcinoma is associated with clinical prognosis

Chinese journal of clinical oncology 2006/vol 3/no.3181-184

128. Tanaka N,Odajima T,et al expression of e cadherin in the process of

lymph node metastasis in oral squamous cell carcinoma 2003;89:

557-63.

129. Bagutti C, Speight PM, et al comparison of integrin, cadherin and

catenin expression in squamous cell carcinoma in oral cavity.j pathol

1998;186;8-16.

ANNEXURE – I

WHO CLASSIFICATION OF ORAL CAVITY AND OROPHARYNX

TUMOURS

Malignant Epithelial tumours

Squamous cell carcinoma

Verrucous carcinoma

Basaloid squamous cell carcinoma

Papillary squamous cell carcinoma

Spindle cell carcinoma

Acantholytic squamous cell

carcinoma

Adenosquamous carcinoma

Carcinoma cuniculatum

Lymphoepithelial carcinoma

Epithelial precursor lesions

Benign epithelial tumors

Papillomas

Squamous cell papilloma and

verruca vulgaris

Condyloma acuminatum

Focal epithelial hyperplasia

Granular cell tumor

Keratoacanthoma

Salivary gland tumors

Salivary gland carcinomas

Acinic cell carcinoma

Mucoepidermoid carcinoma

Adenoid cystic carcinoma

Polymorphous low-grade

adenocarcinoma

Basal cell adenocarcinoma

Epithelial—myoepithelial

carcinoma

Clear cell carcinoma, not otherwise

specified

Cystadenocarcinoma

Mucinous adenocarcinoma

Oncocytic carcinoma

Salivary duct carcinoma

Myoepithelial carcinoma

Carcinoma ex pleomorphic

adenoma

Salivary gland adenomas

Pleomorphic adenoma

Myoepithelioma

Basal cell adenoma

Canalicular adenoma

Ductal papilloma

Cystadenoma

Soft Tissue Tumors

Kaposi's sarcoma

Lymphangioma

Ectomesenchymal chondromyxoid

tumor

Focal oral mucinosis

Congenital granular cell epulis

Hematolymphoid tumors

Diffuse large B-cell lymphoma

(DLBCL)

Mantle cell lymphoma

Follicular lymphoma

Extranodal marginal zone B-cell

lymphoma (MALT) type

Burkitt lymphoma

T-cell lymphoma (including

anaplastic

large cell lymphoma)

Extramedullary plasmacytoma

Langerhans cell histiocytosis

Extramedullary myeloid sarcoma

Follicular dendritic cell

sarcoma/tumor

Mucosal malignant melanoma

Secondary tumours

ANNEXURE II

TNM STAGING OF CARCINOMAS OF ORAL CAVITY AND

OROPHARYNX

PRIMARY TUMOR (T) FOR ORAL CAVITY AND LIP

TX - Primary tumor cannot be assessed

T0 - No evidence of primary tumor

Tis - Carcinoma in situ

T1 - Tumor ≤2 cm in greatest dimension

T2 - Tumor 2 to 4 cm in greatest dimension

T3 - Tumor >4 cm in greatest dimension

T4a (lip) - Tumor invades through cortical bone, inferior alveolar nerve, floor

of mouth, or skin ( chin or nose)

T4a (oral cavity) - Tumor invades through cortical bone, into deep /extrinsic

muscle of tongue (genioglossus, hyoglossus, palatoglossus, and styloglossus),

maxillary sinus, or skin of face

T4b (lip & oral cavity) - Tumor invades masticator space, pterygoid plates, or

skull base and/or encases internal carotid artery

PRIMARY TUMOR (T) FOR OROPHARYNX

TX - Primary tumour cannot be assessed

T0 - No evidence of primary tumour

Tis - Carcinoma in situ

T1 - Tumour 2 cm or less in greatest dimension

T2 - Tumour more than 2 cm but not more than 4 cm in greatest dimension

T3 - Tumour more than 4 cm in greatest dimension

T4a - Tumour invades any of the following: larynx, deep/extrinsic muscle

of tongue (genioglossus, hyoglossus, palatoglossus, and

styloglossus),medial pterygoid, hard palate, and mandible

T4b -Tumour invades any of the following: lateral pterygoid muscle,pterygoid

plates, lateral nasopharynx, skull base; or encases the carotid artery

REGIONAL LYMPH NODES (N)

NX - Regional lymph nodes cannot be assessed

N0 - No regional lymph node metastasis

N1 - Metastasis in a single ipsilateral lymph node, ≤3 cm in greatest dimension

N2a - Metastasis in a single ipsilateral lymph node, 3-6cm in greatest

dimension

N2b - Metastasis in multiple ipsilateral lymph nodes, none >6 cm in greatest

dimension

N2c - Metastasis in bilateral or contralateral lymph nodes, none >6 cm in

greatest dimension

N3 - Metastasis in a lymph node >6 cm in greatest dimension

DISTANT METASTASIS (M)

MX - Distant metastasis cannot be assessed

M0 - No distant metastasis

M1 - Distant metastases

STAGE GROUPING

The overall pathologic AJCC stage is

Stage 0 Tis N0 M0

Stage I T1 N0 M0

Stage II T2 N0 M0

Stage III T1,T2 N1 M0

T3 N0, N1 M0

Stage IVA T1,T2,T3 N2 M0

T4a N0,N1,N2 M0

Stage IVB Any T N3 M0

T4b Any N M0

Stage IVC Any T Any N M1

MASTER CHART

S.NO AGE SEX SITE GRADE LN E CAD VIM

1 31 M Tongue WD neg HS neg

2 60 F Bm MD pos HS neg

3 70 F Bm WD neg LS neg

4 65 F Tongue MD pos LS neg

5 70 F Bm MD pos LS LS

6 57 M Bm WD neg HS neg

7 48 F Bm WD neg HS neg

8 55 F Bm MD pos LS neg



11 60 F Tongue MD pos LS LS


13 65 F Tongue MD neg LS HS





18 57 M Ton-base PD pos neg HS

19 65 F Tongue WD neg HS neg



22 65 F Lip PD pos neg HS

23 65 M Lip WD neg HS neg

24 60 F Tongue WD neg LS HS

25 65 M Phary.wall MD neg neg LS

26 74 F Bm MD pos LS LS

27 73 M Tonsil MD pos neg HS


29 75 F Tonsil MD neg neg LS


31 75 F Tongue MD pos LS HS

32 53 M Tongue PD pos neg HS


34 65 M Ton-base WD neg HS neg

35 67 F Tongue MD neg neg LS

36 55 M Floormouth WD neg neg neg

37 80 M Bm MD pos neg HS

38 70 F Floormouth MD neg neg HS

39 69 M Tonsil MD pos neg LS

40 65 M Softpalate WD neg neg HS

41 60 M Tongue WD neg neg LS

42 51 M Bm MD pos HS neg

43 65 M Bm MD neg neg HS

44 59 M Hardpalate MD pos LS neg

45 45 M Tonsil WD neg HS neg


47 45 M Retromola WD neg HS neg

48 70 M Retromola PD pos neg HS

49 70 F Ton-base PD pos neg HS


M - Male Bm - Buccal Mucosa

F - Female HS - High Score

WD - Well Differentiated LS - Low Score

MD - Moderately Differentiated

PD - Poorly Differentiated

1

INTRODUCTION

Oral cancer is one of the most common cancer worldwide ranking

sixth in line.1 In India it ranks first among males and is the third most common

among females. Malignancies that affect any area in the oral cavity, pharyngeal

regions and salivary glands are collectively called as oral cancer.2 Oral cancer

accounts for 3% approximately of all malignancies in toto3,4

. Oral squamous

cell carcinoma constitutes more than 90% originating from the mucosa that

lines with the neoplasms arising in minor salivary glands and soft tissues, that

are rare.3,4

Squamous cell carcinoma of oral cavity and oropharyngeal region

is considered as an aggressive malignant neoplasm, by means of high

mortality and morbidity, commonly occurring in middle-aged males and

older individuals . The 5-year survival rate for oral carcinomas is about 55%,

despite the therapeutic advances and also considerably reduced for more

posteriorly located tumours.5

Majority of squamous cell carcinoma is unequivocally associated with

tobacco chewing and are preceded by precancerous lesions. Oral squamous cell

carcinogenesis is a multistep process that involves genetic events leading to

modification of the normal function of oncogenes and tumour suppressor

genes2.

The earliest morphologic changes that could be detected are the

appearance of premalignant lesions that includes leukoplakia and

erythroplakia. Fifty Percentage of leukoplakias exhibit dysplasia and the

overall malignant transformation potential is about 0.1-2% peryear.6 The

2

changes of dysplasia is more in erythroplakic lesions than in homogenous

leukoplakia. The chances for simple hyperplastic lesions to turn malignant is

0.9% whereas moderate and severe dysplasia have 16% incidence rate of

malignant transformation.7 Greater the accumulation of mutations in tissue

greater are the chances for malignancy. Death among these patients is mostly

because of regional in cervical lymph nodes and distant metastasis.

A combination of relevant clinical data, adequate sampling, detailed

histopathological examination & techniques like immunohistochemistry are

important to identify the neoplastic lesions with high malignant potential of

invasion and metastasis. Hence detection of high risk cases for distant

metastasis at an early stage is mandatory for the better prognosis of the

individual.

3

AIMS AND OBJECTIVES

1. To identify the incidence and distribution of oral cavity and

oropharyngeal SCC in patients admitted in Karpaga Vinayaga Institute

of Medical Sciences during the year 2015-2016.

2. To identify the clinicopathological features of oral scc including age,

sex, risk factors, tumor location, lymphnode metastasis and grade of

tumor.

3. To study the expression of immunological markers E- cadherin and

vimentin in SCC

4. To evaluate the relation of expression of markers with respect to age,

sex, site, lymph node metastasis and grade of tumor.

5. To assess their role in the determination of invasion and metastasis

6. To help the clinician suggest newer therapeutic markers for planning

further treatment in high risk oral SCC.

4

REVIEW OF LITERATURE

EPIDEMIOLOGY

Oral and oropharyngeal carcinoma is the sixth most common cancer in

the world.1 Amongst the western countries it represents about 2-4% of all

malignancies and in India it accounts about 40% of all malignancies.8,9

Squamous cell carcinoma is the most common type of oral and oro-pharyngeal

cancer, arising from the lining squamous epithelium and this constitutes more

than 90% of oral cancer cases.3-5

Oral and oropharyngeal SCC takes about 3%

and 2% of total malignancies in men and women, respectively.10

As a result of heavier indulgence in tobacco and alcohol habits, a male

predilection was observed in most countries. In India, women showed highest

rates of intraoral malignancy attributable to heavy tobacco chewing. However,

the world wide male to female ratio was lower for oral cavity cancer than

oropharyngeal cancer, possibly explaining that higher exposure to tobacco and

alcohol are required to initiate oropharyngeal cancer.4 Individual assessment

of incidence rates for the cancers of oral cavity and oropharynx is complicated

by the difficulty in assigning a site of origin to tumours that are often

advanced.

MICROANATOMY OF ORAL MUCOSA

The oral cavity is a multifaceted organization tailored to perform

various functions including mastication, ingestion, taste sensation, immune

surveillance and speech. The squamous epithelium lining the oral mucosa

5

is composed of keratinocytes which is stratified. Basal cells are the one that

helps to maintain the normal thickness of the epithelium by their constant

replications. The basal cells are constituted by the organized units of stem cells

and transit amplifying cells. The transit amplifying cells divide frequently in

short intervals whereas the stem cells divide infrequently. The oral squamous

epithelium has a longer turn over time when compared with gastrointestinal

mucosa . It takes about 25 days for buccal epithelium and 50 days for gingival

epithelium.11

Lamina propria that lies beneath the epithelium is composed of

fibrous tissue with very rich neurovascular supply. The crucial factor for the

homeostatic maintenance of oral mucosa is the interface between the

epithelium and lamina propria.

VARIATION IN THE LINING EPITHELIUM IN ORAL SITES

Epithelial type Thickness Site

Orthkeratinised Thick Hard palate, gingiva

Parakeratinised Thick Gingival, dorsal tongue,

alveolar mucosa

Non keratinised Thick Buccal and labial

mucosa

Non keratinised Thin

Ventrolateral tongue,

floor of the mouth, soft

palate and gingival

sulcus

6

ORAL EPITHELIAL CELL KINETICS

The oral epithelial cell proliferation index is the one exhibiting high

labelling index rate of 11.7% in non keratinized lining mucosa than the thick

keratinized gingival mucosa, which showed an index of 8.5%12

.

ETIOLOGY

The etiology of precancerous and cancerous oral lesions are predictably

multifactorial in nature, although the world wide well recognized risk factors

are tobacco use (smoking, snuffing or chewing)and high alcohol intake. The

betelquid use plays a significant role in India and south asian countries.

TOBACCO

In India tobacco chewing accounts for about 50% of oral and

oropharyngeal malignancies in men and about 90% in women.4 The risk of oral

cancer is three and half times higher in smokers when compared to non

smokers 8. International agency for research on cancer (IARC). confirmed that

tobacco in various forms of smoking(e.g., cigars and cigarette, bidis, pipes,) is

carcinogenic in humans.15

Tobacco is known to contain more than 70 known

carcinogens, most important of them being polycyclic hydrocarbons such as

benzo(a) pyrene and nitrosamines. Reactive carcinogenic intermediates are

said to be formed as a result of oxidizing enzyme metabolism by cytochrome

p450. The failure to detoxify these carcinogens leads to the formation of

adducts between the carcinogens and oral keratinocyte DNA, have been

implicated in the mechanism of carcinogenesis.16

7

It was found that, combination of areca nut, calcium hydroxide with

tobacco in betel quid is associated with 8-15 times high relative risk as

compared to that of 1-4 times, without tobacco. The observation was that

reactive oxygen species which are generated during chewing are known to

induce mutations, or by making the mucosa susceptible to environmental

toxicants.

Boyle et al in his study observed the changes in the buccal mucosa of

smokers which showed the over expression of multiple genes in the oral

carcinogenesis, increased prostaglandin levels and langerhan cell population. 17

Risk of oral Cancer is related to both the intensity and duration of

tobacco use with smoking more than 20 cigarettes per day for more than 20

years duration was associated with increased risk of malignancy.18

ALCOHOL

Alcoholic beverages contain variable amounts of ethanol and

carcinogens which include nitrosamine, acrylide and polyphenols 18

. Ethanol

when metabolized to acetaldehyde exerts its mutagenic effects and acts

synergistically with tobacco smoking in the oral carcinogenesis. Alcohol is

known to act as a solvent and enhances the permeability of oral mucosa to

carcinogens.It was found from the study of Goodson et al.,on the relative risk

factors of alcohol and tobacco that 75% of all oral cancers are preventable.19

DIET AND NUTRITION

Vitamin A, Vitamin C, Vitamin E supplements showed a protective role

in oral cancer due to its antioxidant properties, while processed meat products

8

and red chilli were thought to be risk factors. The thickness of the epithelium is

maintained by the dietary iron normally, because deficiency of iron is found to

result in oral epithelial atrophy leading to cancer in upper air and food

passages. It has been estimated that nearly 90% of oropharyngeal cancers can

be prevented by increased consumption of fruits and vegetables. 20

INFECTION

HPV infection constitutes about 40% of oro pharyngeal (especially

tonsil) cancers.2 HPV E6 protein acts by inactivating p53 and HPV E7 protein

inactivates retinoblastoma protein thereby playing a role in the early steps of

oral carcinogenesis2. About a small fraction of HPV-infected lesions require

exposure to chemical carcinogens for their tumorogenic conversion21-25

. Other

infective agents may cause oral cancer are candida albicans and chronic

syphilis.

Other factors implicated in developing oral cancer and its progression

includes ultraviolet radiation (especially in lip cancer), poor oral hygiene,

immunosuppression, periodontal disease, trauma and dental irritation,

xeroderma pigmentosa, Fanconi’s anemia, Bloom syndrome.11

SITES AND INCIDENCE

The incidence of squamous cell carcinoma varies between the different

anatomic sites. The most common site for SCC is lip which is followed by

intra oral sites. The most frequent intra oral sites are the horse shoe shaped area

which includes ventrolateral aspect of tongue, floor of mouth and soft palate

9

complex. In the oropharynx it is the base of the tongue followed by tonsillar

area 4.

MOLECULAR PATHOGENESIS

Oral pathogenesis has been studied in a great detail. It was found that

the relative risk of development of oral cancer in first degree relatives varies

from 1.1-3.8 odds ratio. 51

(CYP1A1 and GSTM1) are few of the genes identified as the genetic

predisposition of OSCC involving xenobiotic metabolism.52,53

Alcohol

dehydrogenase type 3 genotype also is known to be a high risk for the

development of oropharyngeal cancer.54,55

Oral squamous cell carcinoma arises due to multiple factors those

involving accumulation of genetic alterations and acquired due to exposure to

environmental carcinogens such as tobacco, alcohol, micro-organisms,

chemical carcinogens, and ultraviolet or ionizing radiation.56-59

Tumorigenic genetic changes includes two major types: oncogene

activation and inactivation of tumor suppressor genes. Oncogenes are

categorized as growth factor and its receptor (EGF /EGFR), transcription

factors (myc, jun), cell cycle regulators (cyclin D1) and apoptosis inhibitory

factors (bcl2).60

Oncogenes are activated through gene amplification,

augmented transcription, or increased transforming activity due to various

mutations. Inactivation of tumour suppressor genes occurs through various

genetic changes such as mutation, deletion, loss of heterozygosity or by

epigenetic alterations mainly DNA methylation or chromatin alterations.21

10

FIELD CANCERISATION

The theory as defined by field cancerisation states that the entire oral

epithelium is at risk of developing malignancy as a result of constant exposure

to several carcinogenic factors and accumulation of genetic aberrations

affecting the oncogenes and tumor suppressor genes.61

According to this theory various oral cancers develop from independent

cell clones. More recent studies modified this theory into the patch field

carcinoma model.62

This model states that stem cells located in the basal layer

of oral epithelium acquires a genetic aberration which is transferred to its

daughter cells. This patch of cells expands and cannot be seen

macroscopically. In some instance it may present clinically as either

leukoplakia or erythroplakia.

DIAGNOSTIC MODALITIES FOR POTENTIALLY MALIGNANT

DISORDERS

BIOPSY TECHNIQUES

Usually incisional biopsies are performed, except for very small lesions.

The critical issue in incisional biopsy is to obtain tissue of adequate size and

depth including the reticular lamina propria in case of thick keratinizing

lesions. Punch biopsy is preferred as it is better in terms of minimal trauma

and adequate sampling.63

A proper orientation of the specimen is important

otherwise it can exaggerate the complex architecture in the interphase between

the oral epithelium and the lamina propria. In laser excision technique the

entire potentially malignant lesion can be sampled.

11

Other diagnostic methods – Exfoliative oral cytology has high

sensitivity and specificity64,65

in the evaluation of potentially malignant lesions

and identifying dysplasia in these lesions.66

Cytologically dysplasia can be

assessed either in the direct smear or liquid based cytology.However biopsy

has to be performed if dysplasia is identified, as architectural changes and

invasion cannot be recognised in exfoliative cytology.

Classification of oral potentially malignant disorders

Precancerous lesions

Leukoplakia

Erythroplakia

Erythroleukoplakia

Pre cancerous conditions

Oral submucosal fibrosis

Actinic keratosis

Lichen planus

Siderophagic dysphagia

Discoid lupus erythematous

Palatal lesions in reverse cigar smoking

Syphilis

Dyskeratosis congenital

Epidermolysis bullosa

Immunosuppression

12

SQUAMOUS CELL CARCINOMA

Oral cavity and oropharnygeal carcinomas are classified according to

the WHO (Annexure I). More than 90% of the malignancies are squamous cell

carcinoma. The remaining comprises of all other type of SCC which vary

widely in their degree of differentiation. Histologically, conventional SCC is

characterized by arrangement as infiltrating cords and nests of malignant

squamous epithelial cells. The biological behaviour of carcinoma is correlated

with the histological grade to some extent. The histological variation of the

tumor is based on the degree of differentiation and keratin formation.

Perineural and vascular invasion are seen common among these carcinomas.

CONVENTIONAL SCC

SCC varies widely in their degree of differentiation. Histologically,

conventional SCC is characterized by infiltrating cords and nests of malignant

squamous epithelial cells.The epithelium adjacent to the invasive tumour often

exhibits dysplastic changes of varying grades.13

Majority of the tumour show

some lymphocytic response. Some the cases show eosinophilic response

which is said to carry a good prognosis.

VARIANTS

VERRUCOUS CARCINOMA

This type is a well differentiated variant of OSCC with indolent

clinical behavior and excellent prognosis81

. Seventy Five Percentage of

verrucous carcinoma occurs in oral cavity and larynx with most common site

being lower lip and hard palate4. Clinically it presents as cauliflower like

13

exophytic growth with a warty surface. Microscopically, it is characterized by

proliferation of stratified squamous epithelium with bulbous rete ridges

exhibiting mild cytological atypia. The deep surface invaginations are filled

with ortho and parakeratin. Metastasis to distant sites and lymph node

involvement is rare when compared to conventional SCC. 4,18

BASALOID SCC

It is one of the rare aggressive variant, first identified by Wain and

others19

. Its occurrence being common in oropharynx than in oral cavity.

Clinically appears as an ulcerated or an exophytic mass.14

Histologically it

exhibits a tubular or glandular like pattern with central comedo necrosis and

peripheral palisading arrangement. Lack of oral epithelial continuity leads the

diagnosis of poorly differentiated SCC. Early recurrence and local metastasis

is common and has been considered as the worst prognosis than conventional

SCC20.

ACANTHOLYTIC SCC

An uncommon variant, which is characterized by acantholysis within

the tumour cell nests resulting in the glandular or the pseudoluminal

appearance. Most common in the sun-exposed areas of head and neck regions

commonly lips4. Prognosis is similar to conventional SCC but some studies

showed aggressive behavior.for this variant.

14

PAPILLARY SCC

Papillary SCC is rare in oral cavity with larynx and hypopharynx being

the most common sites. It may evolve from pre-exist papillary hyperplasia or

squamous papilloma. Papillary squamous cell carcinoma carries better

prognosis when compared with the other variants4.

SPINDLE CELL CARCINOMA

It is composed of squamous cell carcinoma and malignant spindle cell

component of epithelial origin. It may develop following radiation exposure.15

STAGING

Prognostic evaluation is based mainly on clinical TNM staging

(Annexure II). Recently several studies suggested the limited prognostic value

of conventional TNM staging, as factors like tumour thickness and depth of

invasion which are directly related to prognosis are not included.21,22

According to Woolgar et al., the two well established histological factors

predicting the behaviour are the tumor thickness and extracapsular nodal

spread 22

.

Grading systems

Broder in 1920, developed a quantitative grading of 3 categories based

on the degree of differentiation. In 1973 Jakobsson et al.23

developed a

multifactorial grading system to obtain precise prognostic evaluation which

was later modified by Anneroth and Hansen for the application to squamous

cell carcinoma of tongue and floor of mouth24

. Histological grading based on

Broder’s system has a documented prognostic value, but it has not been

15

incorporated into therapeutic strategies. This is mainly due to the subjective

variation of the current grading system25

.

According to WHO grading system 3 categories are recommended –

well differentiated, moderately differentiated and poorly differentiated16

. It

depends on the subjective assessment of keratinisation, pleomorphism and

mitotic activity18,22

.

Broder’s grading system24

Grade I: Well differentiated = <25% of undifferentiated cells

Grade II: ModeratelyDifferentiated=<50% of undifferentiated cells

Grade III: Poorly differentiated =<75% of undifferentiated cells

Grade IV: Anaplastic or pleomorphic=>75% of undifferentiated cells

Anneroth grading included the six parameters of which 3 are connected

to tumor population and other 3 are connected with tumor host relationship.

The six parameters of Anneroth’s histological grading system included the

degree of keratinitization, nuclear pleomorphism, number of mitoses, pattern of

invasion, stage of invasion, lympho-plasmocytic Infiltration.

16

As proposed by Anneroth et al: Malignancy grading system for oral

squamous cell carcinoma

Morphological

parameter

Points

1 2 3 4

Degree of

keratinisation

Highly keratinised

(50% of the cells )

Moderately

keratinised

(20-50%of the

cells)

Minimally

keratinised

(5-20%of the cells )

No keratinisation

(0-50%of the

cells )

Nuclear

pleomorphism

Little nuclear

pleomorphism

(75% matire cells)

Moderately

abundant nuclear

pleomorphism

(50-75%mature

cells)

Abundant nuclear

pleopmorphism

(25-50% mature

cells )

Extreme nuclear

pleomorphism

(0-25% mature

cells)

No.of

mitosis/HPF 0-1 2-3 4-5 5

Akhter et al., observed that the Anneroth’s classification based on

multifactorial grading is a better predictor of lymph node metastasis26

. In 1989,

it was Bryne who proposed that the invasive tumour front grading system

gave the prognosis better than other areas of the tumour27

.

The histologic pattern often reflects the metastatic potential of the

tumour population. The poorly differentiated tumors have higher probability of

metastatic potential and it is correlated with survival.

17

PROGNOSTIC FACTORS

Location

Lip carcinoma91

has the highest 5 year survival rate of about 90%

followed by anterior tongue, posterior tongue, floor of mouth, tonsil, gingival,

hard palate in the order of decreasing frequency.

Stage

(Annexure II) It is the significant parameter predicting the prognosis.

The recurrence-free 5-year survival rates for stage I, 91.0%; stage II, 77.2%;

stage III, 61.2%; stage IVA, 32.4%; stage IVB, 25.3%; stage IVC, 3.6%.28

Grade

Grading of the deep invasive margins is more important than grading of

the entire tumour in predicting the prognosis.30

Depth of invasion

Desmoplastic response

In lip carcinoma, presence of desmoplasia a predictor of aggressive

behaviour.

Tissue eosinophilia

Eosinophilic infiltration indicates a better Prognosis31

.

Lymph node involvement

It is a key feature in the staging system. Presence of extra capsular

spread is an indicator of decreased survival rate22

18

DNA ploidy

It correlates with tumour grade and an independent prognostic factor

with nondiploid tumors which carries unfavourable prognosis.

HPV 16

This is an indicator of improved survival among patients with

oropharyngeal carcinoma.

p16

It is a surrogate marker of high risk HPV which carries a favourable

Prognosis32

.

IMMUNOHISTOCHEMISTRY

Albert Coons et al in 1941 first labelled antibodies directly with

fluorescent isocyanate. Nakane and Pierce et al in 1966, introduced the indirect

labeling technique in which the unlabelled antibody is followed by second

antibody or substrate. From then there has been various developments in

immunohistochemistry which includes peroxidase – antiperoxidase method

(1970), alkaline phosphatase labeling (1971), avidin biotin method (1977) and

two layer dextrin polymer technique (1993).19

Immunohistochemistry involves two disciplines – immunology and

histology. Immunohistochemistry is one which is used to determine expression

of particular antigen and its microanatomic location in the tissue. IHC uses

antibodies to detect antigenic differences between the cells.These differences

help us to specifically identify the lineage of cell populations and define

biologically distinct population of cells within the same lineage.

19

Antigen retrieval technique were introduced by Shi and associates in

1991. It’s a simple method that involves heating paraffin sections to a high

temperature before IHC staining.The use of antiboby in IHC depends on

sensitivity and specificity of antigen antibody reaction as well on the

hybridoma technique which provides limitless source of highly specific

antibodies.

Detection systems

Antibodies are usually labelled or flagged by some method to permit

visualization – these of which include fluorescent substances, enzymes forming

colored reaction with suitable substrate (light microscopy) or heavy metals

(electron microscopy ).

Methods of IHC

Direct conjugate labelled antibody method

Antibody is attached with a label by chemical means and directly

applied to tissue sections .this is a rapid and easy procedure and involves

detection of multiple antigens which require separate incubation with specific

antibodies.

Indirect sandwich method

Enzymes are labeled with secondary antibody which is produced against

primary antibody. The advantages are increased versatality , high working

dilution of the primary antibody and easy preparation of secondary antibodies

against a primary antibody of different species.

20

Unlabelled antibody methods

Enzyme bridge technique

Here the labelled moiety will be linked to the antigen solely by

immunologic binding.

Peroxidase antiperoxidase method

The principle of the PAP method is similar to that of the enzyme bridge

method. The acronym PAP denotes the peroxidase antiperoxidase reagent that

consists of antibody against the horseradish peroxidase and horseradish

peroxidase antigen in the form of a stable small immune complex. Available

evidence suggests that this immune complex consists of two antibody

molecules and three horseradish peroxidase molecules in the configuration.

The PAP reagent and the primary antibody must be from the same species

(or that taken from closely related species with common antigenic

determinants), whereas the bridge or linking antibody could be derived from a

second species and has specificity against the primary antibody.

Avidin biotin technique

The high affinity between avidin and biotin is used in this technique ;.

Biotin binds to the primary antibody and avidin binds to the enzyme thus

attaching it to the biotinylated antibody. Disadvantage of this procedure is the

presence of endogeneous biotin activity that produces non specific background

staining.

21

Avidin biotin conjugate procedure

Here the primary antibody is added followed by biotinylated secondary

antibody and next preformed complexes of avidin and biotin horse radish

peroxidase conjugate.

Biotin streptavidin system

In this method Streptavidin is used in place of avidin, Streptavidin

complexes are more stable compared to avidin.

Immunogold silver technique

This is used in ultrastructural immunolocalisation. Gold particles are

enhanced by addition of several layers of silver.

Polymeric method

This technique allows the binding of a large number of enzyme

molecules to a secondary antibody via dextran backbone. The advantages of

this technique are increased sensitivity, minimized non specific background

staining and reduction in number of assay steps.

Alkaline phosphatase and anti alkaline phosphatase method

The principles are same as that of PAP method .

Tissue fixation , Processing and antigen retrieval techniques

Tissues for IHC undergo fixation , dehydration and Paraffin embedding.

Fixation

This is a critical step as preservation of morphology is essential for

interpretation. 10 % neutral buffered formalin is used.

22

It has the following advantages:

1. Good morphological preservation

2. Cheap, easily available, penetrates tissues well and sterilizes them.

3. Carbohydrate antigens are better preserved and does not interfere

with the staining process.

The disadvantage of masking antigens during fixation can be overcome

by antigen retrieval technique.

ANTIGEN RETRIEVAL

Antigen retrieval can be done by the following different techniques to

unmask the antigenic determinants of fixed tissue sections.

1. Proteolytic enzyme digestion

2. Microwave antigen retrieval

3. Pressure cooker antigen retrieval

4. Microwave and trypsin antigen retrieval

PROTEOLYTIC ENZYME DIGESTION

Huank et al in 1976 introduced this technique to breakdown formalin

cross linkages and to unmask the antigen determinants. The most commonly

used enzymes include trypsin and proteinase. The disadvantages include over

digestion, under digestion and antigen destruction.

MICROWAVE ANTIGEN RETRIEVAL

This is a new technique that is most commonly used in current practice.

Microwave oven heating involves boiling formalin fixed paraffin sections in

various buffers for rapid and uniform heating.

23

PRESSURE COOKER ANTIGEN RETRIVEL:

Miller et al in 1995 compared and proved that pressure cooking method

had fewer inconsistencies, less time consuming and can be used to retrieve

large number of slides than in microwave method.33

PITFALLS OF HEAT PRETREATMENT

Drying of sections at any stage after heat pretreatment destroys

antigenicity. Nuclear details are damaged in poorly fixed tissues. Fibers and

fatty tissues tend to detach from slides while heating. Not all antigens are

retrieved by heat pretreatment and also some antigens like PGP 9.5 show

altered staining pattern.

THE CONCEPT OF EPITHELIAL MESENCHYMAL TRANSITION

The epithelial mesenchymal transition is the one which has a crucial

role in embryonic development. It is also involved in metastasis and

progression of cancer with both sharing a common molecular basis. Cancer

cells that acquire a mesenchymal phenotype create a favourable

microenvironment for invasion and metastasis. Further it has been stated that

the phenotype of EMT features has lead to chemoresistance that causes

recurrence and metastasis after standard chemotherapeutic treatment. Thus it

has been proven that EMT has been closely involved in the process of

carcinogenesis, invasion, metastasis, recurrence and chemoresistance. The

continued study in the mechanism of cancer pathogenesis has gone a way

ahead in identifying the involvement of cancer stem cells and micro rna in

EMT.

24

The end point of advanced cancer being distant metastasis has been the

cause of death in most cases.34

Multiple steps are hypothesised in the process

of tumor metastasis all of which are required to achieve tumor spreading 36,37

first of all the steps is the escape of tumor cells from the primary tumor site

following which they enter directly or through the lymphatics. Most of the

cancer cells in circulation do undergo apoptosis due to anoikis conditions38

.

Having escaped apoptosis the cancer cells adhere to the endothelium to

extravasate to surrounding tissues. The final step of all these is the distal

colonisation and invasion of cancer cells to grow in the new

microenvironment. phenotype that was defined for embryology , has now

been extended to cancr progression and metastasis.6,40

The mechanism of

EMT has been studied in tumor samples through use of EMT associated

markers such as mesenchymal specific markers (i.e vimentin and fibronectin)

and epithelial specific markers (i.e E-cadherin and cytokeratin) and

transcription factors SNAIL AND SLUG42

INVOLVEMENT OF EMT IN CANCER PROGRESSION

In 1980’s it was the first analyses by Greenberg and Hey who stated

epithelial mesenchymal transition associated changes in cellular phenotype and

mesenchymal state in adult and embryonic epithelia49,50

. There are multiple

steps in the process of EMT.52,53

The first step being disintegration of cell to cell adhesion with the loss

of epithelial marker E – cadherin and gain of mesenchymal marker vimentin.

Followed by which there is change of polarity from baso apical to front rear

25

polarisation. After which the cytoskeleton undergoes remodelling, cell matrix

adhesion alteration. The same fact was proven by studies in 199060

indeed

through EMT related signalling pathways40,54

. Boyer et al who stated in his

study that EMT during development depends on distinct and specific signalling

molecules that are highly controlled specifically and temporarily and that

which do not occur under normal circumstances.55

MOLECULAR MECHANISM OF EMT

EMT as defined by the epithelial loss and a gain of mesenchymal

characteristics at a cellular level that helps in distant metastasis by the cell.

This process involves the cells to become discohesive and also alters the

organisation of the cytoskeleton which switches the polarity from apical basal

to front rear. Through the secretion of lytic proteases they acquire invasive

properties and resistance to senescence and apoptosis. Multiple regulatory

pathways are involved in emt the foremost being the transforming growth

factor beta signalling activity which is found to be enhanced in all

physiological and pathological conditions. Emt is observed in organogenesis,

inflammationand tumor invasion.113

TGF β binding to its cell surface receptors

(type 1- 111) activates the smad family of transcription factors which then

translocates to the nucleus and combines its activity with the snail and twist

family of factors the so called emt master genes.Non smad signalling

molecules downregulates TGFβ and that those progress emt includes rholike

gtp ases ,phosphotidl inositol(pi3k)114

these together mediate trnscriptonal

repression of genes that are involved in cell polarity and cell – cell adhesion.-

26

rho a and e cadherin. 11,12

E cadherin is regulated by the incoming of histone

deacetylases (HDAC) and other repression of e box elements in the E cadherin

promoter which cause condensation of chromatin and transcriptional

repression. N – cadherin is simultaneously expressed which helps in enhancing

adhesion between mesenchymal cells.This is called cadherin switch which is

the hall mark of emt.115

Membranous expression of e cadherin is also repressed in emt via loss

of epithelial specific intermediate filaments keratin. Apart from the e cadherin

claudin and occluding also are repressed. This repression is maintained

throughout the process of emt.

Normally protein complexes like PAR are involved in intercellular

junctions. Hence degradation of the junction causes weakening in the apical

basal polarity cellular phenotype. The TGFβ signalling via MAP-k axis exerts

proproliferative and antiapoptotic effects.114

map-k alone is also known to

induce emt.

After the degradation of cell junction complexes and losing

cohesiveness the mesenchymal like tumor cells are enabled to invade through

the basement membrane in to the underlyting tissue by the secretion of lytic

enzymes such as matrix mettaloproteinases mmp’s. map –k mediated re

organisation of actin cytoskeleton is enhanced by the expression of vimentin.116

The migration and invasion of tumor cells is facilitated by the special cellular

protrusions like filopodia, lamellopodia and invadopodia. filopodia helps as a

guide throught the ECM matrix via the parallel fashion of actin .Lamellopodia

27

act on branching actin filaments and also helps in cell motility.Inavadopodia in

addition to action like lamellopodia also degrades ecm with proteins likw

mmp-1 , mmp-7,mmp-9.This has been linked to emt mechanism,.transcription

factor like twist 1 and loss of e cadherin was studied in colorectal carcinoma.117

Upregulation of Vimentin is required for the further maturationof

invadopodia. TGFβ and MMP clear the way of ECM for migration of tumor

cells. The same is proved in gastric carcinoma for the invasion in lymph

vessels and distant metastasis. Similarly proven in triple negative breast

carcinomas too.mmp induced mechanism of emt includes e cadherin targeting

and also increase in the intracellular level of reactive oxygen species.

After arrival at ecadherin regulation the site of metastasis it seems a

prerequisite for metastatic colonisation that there has to be reversal of emt the

so called mesenchymal epithelial transition MET, where there is a regain of e

cadherin and loss of vimentin.

Apart from TGFβ signalling pathway for emt mechanism HIF α is also

attributed which acts via notch and β catenin pathway. Wnt signalling pathway

is also well known other pathway for emt which inhinits glycogen synthase

kinaseGSK3b observed as increased expression of catenin. In the recent years

small non coding rna s also known to be involved in emt mechanism .post

transcriptional activation of emt master genes e caherin and vimentin helps in

regulation of these micro rna s.114

The characteristic finding in the EMT as

described to the loss of cell – cell adhesion which is represented by diminished

expression of e-cadherin. E cadherin which is a calcium dependent

28

transmembrane glycoprotein which is on most epithelial tissues helps forming

a tight junction between adjacent cells. Then the loss of e cadherin can lead to

tumor progression, metastasis and poorer prognosis in various human

carcinomas43,44,57,58

Studies proved Genetic or epigenetic alterations that

resulted in loss of e cadherin in gastric cancer. Breast cancer lobular type59, 60.

Hypermethylation of E cadherin promoter region was also found in various

human cancer ,leading to loss of e cadherin expression.61,62

It was by Graff et al whon proposed the instability and heterogenisity

during metastatic progression which was dependent on the degree of

methylation of e cadherin promoter region.61

There by this finding suggested

that the loss of e cadherin by methylation in the primary lesion may drive

metastatic progression, indicating that the EMT is involved in cancer

metastasis.

The Tumour Microenvironment is always composed of the extracellular

matrix (ECM), cancer associated fibroblasts, myofibroblasts, immune cells,

and soluble factors required for cancer progression and metastasis. Interaction

of cancer cells among themselves in the tumor microenvironment can induce

EMT by auto/ paracrine secretion of mediators such as growth factors,

cytokines and ecm proteins.54

There were studies which suggested that a tumor

microenvironment may induce or maintain the EMT. Oral Squamous cancer

cells have the ability to directly induce a myofibroblastic phenotype via

secretion of TGF-β.TGF –β signalling which by stromal myofibroblast can

29

induce secretion of hepatocyte growth factor (HGF) which helps promote

cancer cell proliferation and invasion.65

EMT IN THE MECHANISM OF DRUG RESISTANCE

It has been proven that the cells that undergo EMT has developed the

ability to invade and acquire resistance for most of the anticancer drugs by

various stress conditions such as exposure to radiation and hypoxic

conditions.66,67

For example in colorectal carcinoma on chronic exposure to

oxaliplatin resistance was established to migrate and invade with phenotypic

changes resembling emt.like (spindle cell shape, loss of polarity,intercellular

separation and pseudopodia formation)69

Similarly in Pancreatic and Ovarian

cancer there were resistance to Gemcitabine and Paclitaxel with changes in

emt.

Various types of targeted therapy are developed and used against many

carcinomas leading to improved survival rate and clinical outcome.72,73

But

however EMT had been reportedly conferring resistance to these targeted

agents. Thus it has been geting proven that emt is leading to resistance to

multiple drugs and permitting rapid progression of the tumor. Thus thereby

clarifying the correlation between the mechanism of EMT and drug resistance

may help clinicians select an optimal anticancer drug treatment and also

choose the mode for high risk cases of invasion.

30

CLINICAL SIGNIFICANCE OF EMT

Cells that undergo emt phenomenon may gain metastatic potential but

these may account only a small proportion of the total population of tumor

cells. Tumor budding is one which involves single cancer cell or small cluster

of cells at the invasive front of tumor tissues. Cancer cells in tumor buds have

proven for down regulation of E cadherin and increase the expression of

vimentin and also possess the characterists of cancer stem cells.77,78

USE OF IMMUNOHISTOCHEMISTRY IN ORAL SCC

Although the treatment of OSCC is curable in early stages , the

advanced oral squamous cell carcinoma (stage III and IV) still carries a poor

prognosis. The oral squamous cell carcinoma is usually treated by surgery or

radiation, with or without concomitant chemotherapy.81,82,83

As observed the

local or regional relapse in cervical lymph node and distant mts remain the

main cause of death .By using a reliable biomarker the high risk cases that

progrsses for invasion could be identified and that will help to plan a definitive

treatment strategy therby helping to improve the survival rate of the patient. 84

The epithelial to mesenchymal transition which was characterised by

increased expression of e cadherin and decreased vimentin was consistently

proved by various studies.86,87,88

This process was similar to the epithelial–

mesenchymal transition that has been implicated in tissue remodelling, wound

healing,organ development and cancer progression.87-90

The epithelial–

mesenchymal transition is a complex process that has been always the

governing morphogenesis in multicellular organisms and had lead to all the

31

spectrum of cellular changes, including loss of polarity and adhesion, increased

motility, and the acquisition of a mesenchymal phenotype87-90

Numerous

studies of epithelial malignancies have shown that E-cadherin has a

transcriptional and regulatory role in invasion and metastasis and is associated

with a poor outcome.91-100

E CADHERIN

The 120kda protein e –cadherin is a calcium-dependent transmembrane

glycoprotein of the type 1 cadherin superfamily ,which is encoded by the

CDH1 gene that is located on chromosome 16q21,. This is expressed in most

epithelial cells.96

E-cadherin has a major role in establishing itself in the cell

polarity and in maintaining normal tissue architecture. E Cadherin with its

intracellular domain gets its link to the actin cytoskeletonalong with its its

interaction with its cytoplasmic-binding partners, the catenins (α-, β-, and γ-

Catenin).102

The E-cadherin or the β-catenin complex which , therefore,

functions together as a component of adherent cell–cell junctions that promote

cell adhesion.91,102

This E cadherin binding with the protein member of

TCF/LEF-1 family by either its cytoplasmic accumulation or its translocation

to the nucleus has been associated with various physiological and pathological

processes, including tumour progression.103,104

VIMENTIN

Apart from the aberrant expression of E-cadherin/β-catenin complexes,

the association of vimentin expression de novo has been frequently associated

with the metastatic conversion of epithelial cells and tumour invasion.

32

Vimentin is a type 111 intermediate filament that is normally found in

mesenchymal cells.105

Multiple studies have proven that vimentin is also

expressed in migratory epithelial cells that are involved in embryogenesis and

organogenesis, wound healing, or tumour invasion.106-109

In the study by Gilles

et al showed that the β-catenin/TCF pathway upregulates vimentin , which

suggested that this functional regulation of epithelial cells is involved in

invasion and/or migration.110

The histopathological evaluation of oral squamous cell carcinoma may

vary widely within the same tumour from the central area to the invasive area.

Currently studies have proven that the invasive front of the tumor gives the

most useful prognostic information.111,112

Thus the tumor grade at the invasive

front of the squamous cell carcinoina has proven to be of high prognostic

value.84,111,112

Thus the molecular biomarkers used for evaluation of tumor

invasion helps clinicians to decide on the best treatment strategy.. In this study,

we studied the expression of vimentin and E-cadherin/β-catenin expression in

oral squamous cell carcinomas and examined the potential role of the

epithelial–mesenchymal transition in invasiveness through its association with

clinicopathological features and patient outcome.

Studies from Araujo et al proved a relation between the histological

grading of malignancy and the vimentin expression in 43 cases of oral

squamous cell carcinoma .In this study 60.4% of cases expressed vimentin

especially in tumor with highest scores of malignancy.This correlation of

33

vimentin expression in high histological grade of malignancy suggests that

vimentin is an indicator of poor prognosis in oral scc.118

Maria Carmen et al51

did a study to analyse the immunohistochemicl

expression of e cadherin and CD44v6 in 30 cases of oral scc. 15 of lip and 15

of tongue cases respectively. The pattern of expression and the number of

immunopositive cells were evaluated. It was observed that there was no

significant difference in the pattern of expression and its relation to anatomical

location.But for the histological grading there was very low score or negative

expression of e cadherin for higher grades of malignancy and strong positivity

exhibited in well to moderately differentiated malignancies.119

Lai kui et al made a study in 83 oral scc case. Using quantitative

immunohistochemistry the expression of e cadherin ,vimentin and beta catenin

was observed. It was seen that there was high immunoreactive score for well to

moderately differentiated carcinoma and a lower score for poorly differentiated

carcinoma and vice versa for vimentin, where vimentin was expressed more in

poorly differentiated.This study also observed their correlation with the tumor

invasive front, survival and reccurence. It was found that the expression of

vimentin and e- cadherin were associated with survival and were proved to be

independent prognostic factors in univariate and multivariate analysis.

They proved that the combination of the upregulation of vimentin and

aberrant expression of e cadherin in poorly differentiated squamous cell

carcinoma and at the tumor invasive front were proven to be of a very useful

prognostic marker in oral squamous cell carcinoma. 120

34

Jing ping et al in their study of 42 cases of oral scc again proved that

Ecadherin and vimentin positive expression was associated with tumor

metastasis in oral scc. Their study primarily confirmed that EMT phenomenon

existed during the development of scc. Co evaluation of both e cadherin and

vimentin maight be a valuable tool for oscc positive patient outcome.121

Gurkiran et al studied that e cadherin had an important role in cell – cell

adhesion and the motility and its loss has been associated with oscc

progression. They observed the expression of e cadherin in various grades of

malignancy .It was observed that the immunoreactivity of e cadherin was

found to be inversely correlating to the loss of cell differentiation. The

expression of e cadherin decreased significantly in advanced oral scc.

Increased expression in well and moderately differentiated oral scc but was

negative to low in majority of metastatic lymph nodes. 122

Figure – 1

Vimentin control

35

MATERIALS AND METHODS

PATIENTS AND TISSUE SPECIMENS

A total of 50 primary oral squamous cell carcinoma biopsy specimens

from patients diagnosed during jan 2015 to jan 2016 were obtained from the

files of the Department of oral Pathology and the department of Oral

Maxillofacial Surgery, Karpaga Vinayaga Institute of Medical Sciences and

Researche. Detailed clinicopathological information was obtained from patient

records. Haematoxylin and eosin-stained tissue sections were evaluated to

confirm or correct the previous histological diagnoses according to the revised

criteria suggested by the World Health Organization (2005). The specimens

included radical excisions, simple excisions and small biopsies.All the patients

had been surgically treated primarily in our centre.this study protocol was

approved by the ethics committee.

HISTOPATHOLOGY EVALUATION

The histological characteristics of oral squamous cell carcinoma were

classified into well-, moderately, and poorly differentiated groups (G1–G3) as

per the criteria that was proposed by the World Health Organization.

The clinical staging and TNM classification were determined as per the

the International Union Against Cancer tumour classification for each patient:

the T was classified into the 4 -T1, T2, T3, and T4 categories and the N

classification as into lymph node-negative (N0) and lymph node-positive (N)

categories. The stage grouping was further divided into the stage I, I, III, and

IV categories.

36

SOURCE OF DATA

A total of 50 cases of oral and oropharyngeal squamous cell carcinoma

sent to department of pathology ny oromaxillofascial surgery unit were chosen

for the study.

INCLUSION CRITERIA

All patients diagnosed to have the squamous cell carcinoma in oral

cavity and oropharynx irrespective of age, sex, clinicopathological

characteristics were included in this study.

EXCLUSION CRITERIA

1. Benign lesions like papilloma and other non neoplastic lesions

2. Carcinomas other than squamous cell carcinoma

3. Cases with inadequate material.

THE PATTERNS OF INVASION

The patterns of invasion of tumor was examined at the normal tissue–

tumour interface. As per Bryne’s claasification the various patterns were

divided in to four types.6,35,36

Type 1 Pattern of invasion represents the tumour

invasion in a broad pushing manner with a well-delineated infiltrating border.

Type 11 is were it has broad pushing ‘fingers’ or separate large tumour islands,

with a stellate appearance.type 111 invasion as invasive tumour islands greater

than 15 cells per island. Type iv Pattern of invasion represents invasive islands

of tumour smaller than 15 cells per island, including cord-like and single-cell

invasions.

37

METHOD OF TISSUE PREPARATION FOR IHC

10% buffered formalin was used to fix the tissues. Then the tissues

were processed in various grades of alcolhol and xylol using automated

histokinette.Paraffin blocks were prepared and sections of 5 micron thickness

were cut and stained using H& E. Suitable blocks were chosen for IHC.

Sections for immunohistochemistry were also cut in the semi automated

microtome. These sections were 4 microns thick and positively charged slides

were used. Sections were subjected to antigen retrieval technique by pressure

cooker method using TRIS EDTA (pH 9) buffer solution and then treated by

HRP ( horse radish peroxidase ) polymer technique.

HRP POLYMER TECHNIQUE

1. The sections were deparrafinised in xylene or xylene substitutes

2. Rehydrated through graded alcohols

3. The slides were then washed in running tap water

4. The antigen retrieval was performed using the appropriate buffer by

pressure cooker method.

5. The endogeneous peroxide was blocked using peroxidase block for 5

mins

6. Slides were then washed in 2 changes of TBS buffer for 5 mins each.

7. Slides were then incubated with protein block for 5 mins

8. Then slides were washed in 2 changes of TBS buffer for 5 mins

each.

38

9. Optimally diluted primary antibody was then used to incubate the

slides for 60 mins.

10. Then the slides were washed in 2 changes of TBS buffer for 5 mins

each.

11. Then incubation with post primary for 30 mins


each.

13. Then incubation with novolink polymer for 30 mins


each.

15. Then peroxidase activity was developed with DAB working solution

16. The slides were then rinsed in water, counterstained in hematoxylin

washed in water, dehydrated , cleared and mounted to be examined.

Normal oral mucosal tissues and breast tissue were used as a positive

control and Negative controls were included in each slide run (omission of

primary and secondary antibodies), and all controls gave appropriate results.

EVALUATION OF IMMUNOREACTIVITY

A semiquantitative evaluation was done which was based on the

staining , the intensity and the distribution using the immunoreactive score (37,

38) which combined the intensity and proportion score. (Immunoreactive

score=intensity score × proportion score). The intensity score was defined as 0

as negative; 1= weak; 2= moderate; or 3= strong, and the proportion score was

defined as 0, negative; 1= <10%; 2= 11–50%; 3= 51–80%; or 4,>80% positive

39

cells. The total score was totally from 0 to 12. Based on the final score The

immunoreactivity was further divided into three groups : negative

immunoreactivity was defined as a total score of 0, low immunoreactivity was

defined as a total score of 1–4, and high immunoreactivity was defined as a

total score >4. The immunostaining of the tumour invasive front was evaluated

using the same method. The stained tumour tissues were scored, although

blinded to the clinical patient data.

40

OBSERVATION AND RESULTS

Expression of E-cadherin and vimentin in oral squamous cell carcinoma

patients

E cadherin : it was observed mainly on the cell membrane of the basal

layer of the spinosum layers. It has a distinct membranous expression.

Vimentin was seen in the cytoplasm of the connective tissue

mesenchymal cells of the normal oral mucosal tissue but not in the normal

squamous epithelium.

All the 50 cases studied were subjected to immunohistochemical study

for E-cadherin and vimentin:

E CADHERIN

Total cases =50

Total cases positive for e cadherin =34(68%)

No.of cases which showed high expression =26(76%)

No.of cases which showed low expression =8(23.5%)

Cases negative for e cadherin = 16(32%)

VIMENTIN

Total cases =50

No.of cases positive for vimentin =16(32%)

No.of cases with high expression = 9(56%)

No.of cases with low expression =7(44%)

Cases negative for vimentin =34(68%)

41

The clinicopathologial variables were compared with the expression of e

cadherin and vimentin separately and their p value was assessed .

TABLE 1

AGE WISE DISTRIBUTION OF SQUAMOUS CELL CARCINOMA

Age Distribution NO.OF CASES %

≤ 40 years 3 6.00

41-50 years 8 16.00

51-60 years 16 32.00

61-70 years 18 36.00

71-80 years 5 10.00

Total 50 100

CHART 1 AGE DISTRIBUTION

In our study the SCC had a peak incidence in the age group of 61-70 yrs

which had 18 out of 50 cases(36%), and 16 (32%) were in the age group of 51-

60 yrs.The youngest age of presentation observed was 31 yrs .

3, 6%

8, 16%

16, 32%

18, 36%

5, 10%

≤ 40 years

41-50 years

51-60 years

61-70 years

71-80 years

42

The mean age was 55yrs. This observation is in concurrence with the

study done by Shubha et al (124),who observed the mean age of 60yrs with

range from 30-70 yrs . The IHC of e cadherin interpretation was done and

assessed age wise

GRAPH 1 AGE WISE DISTRIBUTION OF E CADHERIN

TABLE 2

Age Distribution

(E-cadherin) E-cadherin +ve % E-cadherin -ve % Combined %

≤ 40 years 3 8.82 0 0.00 3 6.00

41-50 years 8 23.53 0 0.00 8 16.00

51-60 years 12 35.29 4 25.00 16 32.00

61-70 years 9 26.47 9 56.25 18 36.00

71-80 years 2 5.88 3 18.75 5 10.00

Total 34 100 16 100 50 100

3

8

12

9

2

0 0

4

9

3

0

2

4

6

8

10

12

14

≤ 40 years 41-50 years 51-60 years 61-70 years 71-80 years

Nu

mb

er

of

Sub

ject

s

Age Distribution (E-cadherin)

E-cadherin +ve E-cadherin -ve

43

TABLE 3

Age Distribution

(E-cadherin) E-cadherin +ve E-cadherin -ve

N 34 16

Mean 55.79 66.19

SD 10.53 7.28

p value

Unpaired t Test 0.0009

Out of the 18 cases in the age group of 61-70 yrs 9 were e cadherin

positive (26.4%) .the p value was turning out significant of 0.0009 in our

study which was to consider an association between advancing age group with

increased incidence of oral squamous cell carcinoma and their positivities to e

cadherin.

GRAPH 2 OBSERVATIONS FOR VIMENTIN

0 0

5

11

5

3

8

11

7

00

2

4

6

8

10

12

≤ 40 years 41-50 years 51-60 years 61-70 years 71-80 years

Nu

mb

er

of

Sub

ject

s

Age Distribution (Vimentin)

Vimentin +ve Vimentin -ve

44

TABLE 4

Age Distribution (Vimentin) Vimentin +ve % Vimentin -ve %

≤ 40 years 0 0.00 3 10.34

41-50 years 0 0.00 8 27.59

51-60 years 5 23.81 11 37.93

61-70 years 11 52.38 7 24.14

71-80 years 5 23.81 0 0.00

Total 21 100 29 100

TABLE 5

Age Distribution

(Vimentin) Vimentin +ve Vimentin -ve

N 21 29

Mean 67.05 53.38

SD 6.65 9.39

P value

Unpaired t Test <0.0001

Out of 18 cases in the age group of 61-70 yrs , 11 cases were vimentin

positive (52.38%) and 5 cases (23.8%)in the age group 51-60 yrs were

positive for vimentin. The mean value for vimentin positivity was 67.05 and p

value which was significant as e cadherin which signified that with increased

age and the positivity for oscc there was increased positivity for vimentin also.

45

TABLE 6

CORRELATION OF E CADHERIN AND VIMENTIN WITH AGE

AGE E CADHERIN

(+ve) (-ve)

VIMENTIN

(+ve) (-ve)

<50 YRS 11 0 0 11

>50YRS 23 16 21 18

p value <0.0009 <0.0001

It was found that there were 23 and 21 cases of e cad and vim positive

beyond the age group of 50 yrs and their p values which were statistically

significant indicated the more risk of scc and their invasion as age advances.

CHART 2 GENDER DISTRIBUTION OF SCC

27, 54%

23, 46%

Gender Status

Male

Female

46

Table 7: SEX DISTRIBUTION OF SCC

Gender No.of cases %

Male 27 54

female 23 46

Of the total 50 cases , 27 cases(54%) were reported in males and

23(46%) in females. Patel MM et al(125) in his study found that the incidence

of OSCC in men and women as 75 % and 25% respectively. Mehrotra rain et al

(126)observed a male to female ratio of 3.27:1 .In concurrence with the above

studies a significant male predominance of 54% was observed in our

study.The similar sex distribution was observed in many similar studies.

GRAPH 3 OBSERVATION OF E CADHERIN ON GENDER

16

18

11

5

0

2

4

6

8

10

12

14

16

18

20

Male Female

Nu

mb

er

of

Sub

ject

s

Gender Status (E-cadherin)


47

TABLE 8 OBSERVATION OF GENDER WITH E CADHERIN

Gender Status

(E-cadherin) E-cadherin +ve % E-cadherin -ve %

Male 16 47.06 11 68.75

Female 18 52.94 5 31.25

Total 34 100 16 100

p value

Fishers Exact Test 0.2252

Out of the 27 males 16 were e cadherin positive (47.06%) and 18

among females were positive. The p value was not significant

GRAPH 4 OBSERVATION OF VIMENTIN

1011

17

12

0

2

4

6

8

10

12

14

16

18

Male Female

Nu

mb

er

of

Sub

ject

s

Gender Status (Vimentin)


48

TABLE 8

Gender Status

(Vimentin) Vimentin +ve % Vimentin -ve % Combined %

Male 10 47.62 17 58.62 27 54.00

Female 11 52.38 12 41.38 23 46.00

Total 21 100 29 100 50 100

p value


Among the 27 males 10 were vimentin positive (47.6%) and 11 among

23 females (52.3%) were positive. The p value turned out to be 0.5675 which

was not significant.

Table 9 CORRELATIONS OF E CADHERIN AND VIMENTIN WITH

SEX

Sex E cad +ve E cad -ve Vimentin +ve Vimentin -ve

Male 16 11 10 17

female 18 5 11 12

p value 0.2252 0.5675

On observation both e cadherin and vimentin showed positivity with

male predominance and their p value by fischer exact test being significant.

49

TABLE 10: DISTRIBUTION OF SITE OF INVOLVEMENT IN SCC

Tumor Location (E-cadherin) No.of cases %

Tongue 18 36.00

Buccal Mucosa 16 32.00

Lip 2 4.00

Tongue - Base 3 6.00

Tonsil 4 8.00

Retromolar 2 4.00

Hard palate 1 2.00

Soft Palate 1 2.00

Floor of Mouth 2 4.00

Pharyngeal wall 1 2.00

Total 50 100

TABLE 11

Tumor location Shubha bhat et al Patel MM et al Current study

Tongue 21% 23.2% 36%

Buccal mucosa 22% 10% 32%

Tongue base 10% 19.6% 6%

Tonsil 9% 11.3% 8%

In our study 18 cases (36%) were found to involve the

tongue,16(32%)cases involved the buccal mucosa followed by other sites. This

observation is similar to a study which has been done by Shubha et al124

and

Patel MM et al 125.

50

CHART 3 TUMOR LOCATION

It was explained due to the possibility of longer duration of exposure to

tobacco, smoking and alcohol, the most common clinical presentation observed

in SCC was ulcer growth(84%),followed by dysphagia, speech difficulty and

leukoplakia. Shubha et al and Durazzo et al found oral lesions in 71% and 88%

of patients in their study respectively.

36%

32%

4%

6%

8%

4%

2%2%

4% 2%

Tongue

Buccal Mucosa

Lip

Tongue - Base

Tonsil

Retromolar

Hard palate

Soft Palate

Floor of Mouth

Pharyngeal wall

51

GRAPH 5 OBSERVATION OF E CADHERIN DEPENDING ON THE

SITE OF TUMOR

Majority of the studies showed higher incidence in anterior anatomical

regions (tongue, buccal mucosa, base of tongue and alveolus).

15

14

1

1

1

1

1

0

0

0

3

2

1

2

3

1

0

1

2

1

0 2 4 6 8 10 12 14 16

Tongue

Buccal Mucosa

Lip

Tongue - Base

Tonsil

Retromolar

Hard palate

Soft Palate

Floor of Mouth

Pharyngeal wall

Number of Subjects

Tumor Location (E-cadherin)

E-cadherin -ve E-cadherin +ve

52

Table 12

Tumor

Location

(E-

cadherin)

E-

cadherin

+ve

%

E-

cadherin

-ve

% Combined %

P value

Fishers

Exact

Test

Tongue 15 44.12 3 18.75 18 36.00 0.1171

Buccal

Mucosa 14 41.18 2 12.50 16 32.00 0.0554

Lip 1 2.94 1 6.25 2 4.00 0.5421

Tongue –

Base 1 2.94 2 12.50 3 6.00 0.2367

Tonsil 1 2.94 3 18.75 4 8.00 0.0906

Retromolar 1 2.94 1 6.25 2 4.00 0.0906

Hard palate 1 2.94 0 0.00 1 2.00 >0.9999

Soft Palate 0 0.00 1 6.25 1 2.00 0.3200

Floor of

Mouth 0 0.00 2 12.50 2 4.00 0.0980

Pharyngeal

wall 0 0.00 1 6.25 1 2.00 0.3200

Total 34 100 16 100 50 100

Among the 18 cases of scc that occurred in the tongue 15 were

(44.12%) positive for e caherin and 14 (41.18%) of buccal mucosa scc were

positive.

53

GRAPH 6 TUMOR LOCATION (VIMENTIN)

It was interpreted that vimentin positivity was observed more in

carcinomas that was located in the tongue followed by buccal mucosa.

7

4

1

2

3

1

0

1

1

1

11

12

1

1

1

1

1

0

1

0

0 2 4 6 8 10 12 14

Tongue

Buccal Mucosa

Lip

Tongue - Base

Tonsil

Retromolar

Hard palate

Soft Palate

Floor of Mouth

Pharyngeal wall

Number of Subjects

Vimentin -ve Vimentin +ve

54

TABLE 13

Tumor

Location

(Vimentin)

Vimentin

+ve %

Vimentin

-ve % Combined %

P value

Fishers

Exact

Test

Tongue 7 33.33 11 37.93 18 36.00 0.7742

Buccal

Mucosa 4 19.05 12 41.38 16 32.00 0.0269

Lip 1 4.76 1 3.45 2 4.00 >0.9999

Tongue –

Base 2 9.52 1 3.45 3 6.00 0.5650

Tonsil 3 14.29 1 3.45 4 8.00 0.2966

Retromolar 1 4.76 1 3.45 2 4.00 >0.9999

Hard palate 0 0.00 1 3.45 1 2.00 >0.9999

Soft Palate 1 4.76 0 0.00 1 2.00 0.4200

Floor of

Mouth 1 4.76 1 3.45 2 4.00 >0.9999

Pharyngeal

wall 1 4.76 0 0.00 1 2.00 0.4200

Total 21 100 29 100 50 100

Among the 18 cases of scc in tongue 7(33.3%) were positive for

vimentin and 4 (19.05%) of buccal mucosa were positive, followed by tonsil

constituting 14.29%.

55

CHART 4 DISTRIBUTION OF GRADES OF SCC

TABLE 14

Tumor Grade No.of cases %

Well Differentiated 27 54.00

Moderately Differentiated 18 36.00

Poorly Differentiated 5 10.00

Total 50 100

WHO grading system categorizes SCC in to well , moderately and

poorly differentiated based on their degree of differentiation.Tumor grading is

a good predictor of nodal metastasis and recurrence.

27, 54%18, 36%

5, 10%

Tumor Grade

Well Differentiated

Moderately Differentiated

56

FIGURE – 1

Well differentiated Squamous Cell carcinoma

Among the 50 cases 27(54 %) were well differentiated, 18(36%) were

moderately differentiated and 5(10%) were poorly differentiated.

57

GRAPH 7 OBSERVATION OF E CADHERIN ON TUMOR

GRADE

TABLE 15

Tumor Grade

(E-cadherin)

E-

cadherin

+ve

%

E-

cadherin

-ve

% Combined %

Well Differentiated 24 70.59 3 18.75 27 54.00

Moderately

Differentiated 10 29.41 8 50.00 18 36.00

Poorly Differentiated 0 0.00 5 31.25 5 10.00

Total 34 100 16 100 50 100

P value


24

10

0

3

8

5

0

5

10

15

20

25

30

Well Differentiated Moderately Differentiated Poorly Differentiated

Nu

mb

er

of

Sub

ject

s

Tumor Grade (E-cadherin)


58

24 (70.5%)out of 27 cases of well differentiated tumors were e

cadherin positivity and 10(29.4%) of modereately were positive and all 5

cases of poorly differentiated were e caherin negative .the p value was

significant of about 0.0002 to prove the association of e acdherin negativity

in poorly differentiated tumors.

FIGURE – 2

Well differentiated Squamous Cell carcinoma E – Cadherin strong

membranous positivity

59

GRAPH 8 TUMOR GRADE (VIMENTIN)

TABLE 16

Tumor Grade

(Vimentin)

Vimentin

+ve %

Vimentin

-ve % Combined %

Well Differentiated 3 14.29 24 82.76 27 54.00

Moderately

Differentiated 13 61.90 5 17.24 18 36.00

Poorly Differentiated 5 23.81 0 0.00 5 10.00

Total 21 100 29 100 50 100

p value

Fishers Exact Test <0.0001

Vimentin positivity of about 61.9% is observed in moderately

differentiated and 23.8% in poorly differentiated compared to only 14% in well

3

13

5

24

5

00

5

10

15

20

25

30

Well Differentiated Moderately Differentiated Poorly Differentiated

Nu

mb

er

of

Sub

ject

s

Tumor Grade (Vimentin)


60

differentiated and the p value becoming significant to prove the association of

vimentin positivity with advanced stage of tumor.

FIGURE – 3

Well differentiated Squamous Cell carcinoma vimentin negativity

61

TABLE 17 : COMPARISON OF E CADHERIN AND VIMENTIN WITH

GRADE OF TUMOR

Degree of

differentiation E cad +ve Ecad -ve Vim +ve Vim -ve

Well

differentiated 24 3 3 24

Moderately

differentiated 10 8 13 5

Poorly

differntiated 0 5 5 0

p value 0.0002 <0.0001

The P – Value for both e – Cadherin and vimentin were found to be

significant with increased expression of e cad in well differentiated and

vimentin in poorly differentiated tumors.

FIGURE – 4

Moderately differentiated Squamous Cell carcinoma

62

CHART 5 DISTRIBUTION OF CASES AS PER LYMPH NODE

STATUS

TABLE 17

Lymphnode status No.of cases %

Present 17 34

absent 32 66

Among the 50 cases 17 (34%) had lymphnode metastasis and 32(66%)

had no lymph node metastasis.

34%

66%

Lymph Node Metastasis

Positive

Negative

63

GRAPH 9 OBSERVATION WITH E CADHERIN

TABLE 18

Lymph

Node

Metastasis

(E-

cadherin)

E-

cadherin

+ve

%

E-

cadherin

-ve

% Combined %

Positive 9 26.47 8 50.00 17 34.00

Negative 25 73.53 8 50.00 33 66.00

Total 34 100 16 100 50 100

p value


Among the 17 cases that had lymph node metastasis only 9 (52%)

were positive for e cadherin whereas 25 (73.3%) of lymphnode negative

cases were e cadherin positive.

9

25

8 8

0

5

10

15

20

25

30

Positive Negative

Nu

mb

er

of

Sub

ject

s

Lymph Node Metastasis (E-cadherin)


64

FIGURE – 5

Poorly differentiated Squamous Cell carcinoma

TABLE 19

Lymph Node

Metastasis

(Vimentin)

Vimentin

+ve %

Vimentin

-ve % Combined %

Positive 12 57.14 5 17.24 17 34.00

Negative 9 42.86 24 82.76 33 66.00

Total 21 100 29 100 50 100

p value


The P – Value for both the markers combined for cases with lymph

node positivity was significant.

65

GRAPH: 10 LYMPH NODE METASTASIS (VIMENTIN)

Vimentin positivity of about 57% was found in consistent with patient

who had lymph node metastasis and only 9(42%) being positive with

lymphnode negativity.p value also being significant proving the correlation of

metastasis and vimentin positivity

Figure – 6

Poorly differentiated Squamous Cell carcinoma

strong vimentin positive

12

9

5

24

0

5

10

15

20

25

30

Positive Negative

Nu

mb

er

of

Sub

ject

s


66

TABLE 20: EXPRESSION OF E CAD AND VIMENTIN WITH LYMPH

NODE METASTASIS

Ln mts E cad +ve Ecad -ve Vim +ve Vim -ve

Yes 9 8 12 5

no 25 8 9 24

p value 0.1211 0.0039

The P – value for vimentin with lymph node positivity was highly

significant when compared to E – Cadherin.

GRAPH 11 E-CADHERIN VS VIMENTIN

6

28

15

1

0

5

10

15

20

25

30


Nu

mb

er

of

Pat

ien

ts


67

TABLE 19

E-cadherin Vs

Vimentin

Vimentin

+ve %

Vimentin

-ve %

E-cadherin +ve 6 28.57 28 96.55

E-cadherin -ve 15 71.43 1 3.45

Total 21 100 29 100

p value

Fishers Exact Test <0.0001

There were 6 cases (28.5%) that were e cadherin and vimentin positive

and 15 cases (71.43%) were e cadherin negative and vimentin positive .the p

value calculated as per fischer exact test of <0.0001 was significant in

correlating between the e cadherin expression and vimentin in scc patients.

GRAPH 12 E-CADHERIN VS VIMENTIN - METASTASIS POSITIVE

4

5

8

00

1

2

3

4

5

6

7

8

9


Nu

mb

er

of

Pat

ien

ts


68

TABLE 21

E-cadherin Vs

Vimentin -

Metastasis

Positive

Vimentin +ve % Vimentin -ve %

E-cadherin +ve 4 33.33 5 100.00

E-cadherin -ve 8 66.67 0 0.00

Total 12 100 5 100

p value by fischer exact test 0.0294

Among the 17 lymphnode metastasis positive cases 4 cases (33.33%)

were e cadherin and vimentin positive and 8 cases were (66.67%) e cadherin

negative and vimentin positive . The significant p value also correlates the e

cadherin and vimentin expressions in cases that had lymph node

positivity.similar results were found in a study by Tanaka et al(128) who

observed a significant relationship between reduced e cadherin and

invasiveness of OSCC.Bagutti et al (129)showed that that all least

differentiated tumors showed a reduced expresiion of e cadherin in advanced

stages and these tumor cells are said to acquire invasive phenotype.

69

DISCUSSION

The main observations of this study were that well differentiated OSCC

expressed E-cadherin often as strongly as normal stratified squamous

epithelium,while in poorly differentiated oscc expression was less intense or

lost and in moderately differentiated tumor it was expressed in a heterogenous

fashion. E cadherin an important suppressor molecule of tumor development

which functions by enhancing adhesion between the cells and thereby

inhibiting the cellular proliferation and growth of tumors. The human E-

cadherin gene located at chromosimme 16q22.1.The relationship is that down

regulation of e cadherin expression weakens adhesion between cells that is

followed by infiltration , dissemination and metastasis.

1. Comparison of age distribution with e cadherin and vimentin

The age group of patients included in our study varied from less than

40yrs to more than 80 yrs with most patients belonging to the age group of

61-70 yrs. 18 out of 50 cases contributing to 36% belong to the age group 61-

70 yrs and the mean age being 56.99 yrs.out of 18 cases 9 were e cadherin

positive (26.47%) and 9 were e cadherin negative. 11 cases (52.38%) were

vimentin positive in the age group 61-70yrs. number of positive cases

increased as the age advances.

Jingping et al122

in his study in 2015 compared age group with

e cadherin and vimentin positivity and found that most patients aged more than

50 yrs were e cadherin and vimentin positive. The p values calculated were not

significant for any association of age group and marker positivity.

70

Jianming et al127

in 2006 also proved that 15 out of their 43 patients

belonging to the age group more than 60 yrs and on comparing with

e - cadherin positivity the p value was not significant (p=0.084)

TABLE 22

Name of the

study Age group E cad +ve Vim +ve P value

Jing ping

et al >50 yrs 20 14

0.000

0.177

Jianming

zhang et al >60 yrs 15 - 0.084

Present study 61-70 yrs 9 11 0.0009

0.001

2.Comparison of gender with e cadherin and vimentin

In the present study 27 out of 50 cases were males and 23 were females.

Out of the 27 males 16 were e cadherin positive and 10 were vimentin positive.

Among the 23 females 18 were cadherin positive and 11 were

vimentinpositive. No.of positive cases were more among males.but however

on comparing gender with e cadherinand vimentin positive, The p value was

not statistically significant. (0.225 for e cad and 0.5675 for vimentin).

Jingping et al122

in his study compared gender with e cad and vim

positive where they had 26 out of 42 cases with e cad potivity and 16 of

vimentin positivity.Their p value proved statistically insignificant p value of

0.627 each.

Study by jianming zhang et al127

also disproved the significant

association between gender and ecad positivity with p value of 1.356.

71

TABLE 23

GENDER Jingping et al Zhang et al Present Study

Male 23 15 27

Female 19 6 23

E cad +ve 26 15 34

Vim +ve 16 6 21

TABLE 24

Comparison p values

Jing Ping et al Zhang et al Present Study

E cad 0.627 0.084 0.225

Vim 0.627 - 0.5675

3.Comparison of tumor location with e cadherin and vimentin positivity

In the present study out of 50 cases 18 were in the tongue and 16 in

buccal mucosa. Among the 18 cases 15(83%) was e cad positive and 7(39%)

was vimentin positive in the tongue whereas in buccal mucosa 4 was vimentin

positive and 14 was e cadherin positive. The p values were not significant.

When compared with other studies Jingping et al122

who had 16 out of

23 cases for e cadherin positivity and 9 out of 23 cases for vimentin positivity

and their p value is not significant

72

TABLE 25

Marker

expression

Tumor location

Tongue Buccal mucosa

Jing Ping

et al

Present

Study

Jing Ping

et al

Present

Study

E cad +ve 16 15 3 14

-ve 7 3 4 2

Vim+ve 9 7 2 4

-ve 14 11 4 12

TABLE 26

Comparison of p values

P value Jing ping et al Present study

E cad 0.678 0.117

Vim 0.841 0.774

4.Comparison with grade of differentiation

In the current study 27(54%) cases out of 50 were well differentiated

out of which 24 were e cadherin positive and 3 were vimentin positive among

the 18 moderately differentiated 10 were e cad positive and 13(61.9%) were

vimentin positive and among the 5 poorly differentiated cases all 5 were

negative for E - cadherin and positive for vimentin. Contributing to a p value

of 0.0002 which was statistically significant.

On comparing with the study by Jing ping et al 122

who had 42 cases out

of which 9 were poorly differentiated out of which 6(66%) were vimentin

73

positive and only 3 were e cad positive with p value of 0.022 and 0.064. This

study proves that there is a significant association between the higher grades of

tumor and the expression of markers. The positive expression of vimentin and

loss of e cadherin is favouring towards the poorly differentiated and more of

invasion and risk of metastasis.

TABLE 27

E cadherin No. of Cases

Jingping et al Zhang et al Current study

Well differentiated 10 11 24

Moderately

differentiated 13 9 10

Poorlydifferentiated 3 1 0

Vimentin

Well differentiated 3 - 13

Moderately

differentiated 7 - 3

Poorly

differentiated 6 - 5

TABLE 28

P value Jingping et al Zhang et al Current study

E cad 0.022 0.877 0.002

Vim 0.064 - <0.0001

74

5.Comparison with lymphnode metastasis

In the present study out of 17 casees that had lymphnode metastasis

9(26.47%) were e caherin positive and 12 (57.14%) were vimentin positive.p

value was 0.1211 for e cadherin and 0.0039 for vimentin that was significant

henceforth proving the association between the lymphnode status and vimentin

expression.

Studies by Jingping et al122

had 5 cases of e cad positivity and 11 cases

of vimentin positivity among 17 of lymphnode positive cases in total. Jiang

zhang et al 127

in his study 8 out of 24 lymphnode positive cases positive for e

cad and 16(66.67%) negative.

TABLE 29

Marker Jing ping et al Zhang et al Current study

E cad positive 5 8 9

Negative 12 16 8

Vim positive 11 - 12

negative 6 - 5

Marker Jing ping et al Zhang et al Current study

p values - - -

E cad 0.000 0.023 0.1211

Vim 0.003 - 0.0039

Thus all the studies had proved the statistical association for the

lymphnode metastasis showing increased expression of vimentin and decreased

expression of e cadherin.

75

SUMMARY

The concept of EMT is a valuable model for the morphologic and

molecular changes that are observed in tumor cell invasion and tissue fibrosis.

The association between emt like cellular phenotype as shown by changes in

marker protein expression and tumor aggressiveness has been well proven .

This study was carried out in the Department of Pathology in Karpaga

Vinayaga Institute of Medical Sciences and Research in collaboration with

department of oromaxillo fascial surgery. Total of 50 cases of oral squamous

cell carcinoma cases were chosen for the study and IHC markers E cadherin

and vimentin expressions were studied.

The expression of e cadherin and vimentin has significant correlation

with the advanced stages of the tumor and lymphnode metastasis.

In this study a correlation was made with the advancing age group

and the expression of e cadherin and vimentin

There is no significant statistical association between the gender ,site

of tumor and the expression of markers e cadherin and vimentin.

The positivity of E- cadherin was found to be more in well and

moderately differentiated

The expression of E -cadherin was decreased or absent in poorly

differentiated tumors and

The expression of e cadherin was negative or lost in those tumors

with invasive nature and positive for lymphnode metastasis.

76

The expression of vimentin was statistically significant with

increased positivity in poorly differentiated tumors and in those

tumors with the lymph node metastasis .

This study highlighted the role of expression of e-cadherin and

vimentin where decreased or loss of e-cadherin and increased

vimentin expression proved the predictor of high risk cases for

invasion and distant metastasis in oral squamous cell carcinoma

patients who can be picked by clinician for further follow up and

targeted therapy.

77

CONCLUSION

Oral squamous cell carcinoma is one of the most common cancer world

wide and ranks first among the Indian males. It accounts for high morbidity

and mortality despite the improvement in surgical treatment and adjunctive

therapy. The invasion and regional metastasis has resulted in reduced

recurrence free and disease free survival significantly. This necessitates the use

of molecular markers to identify the high risk cases for invasion and

metastasis. The aim of the study was to there by make an attempt to identify

the risk for invasion and metastssis in the diagnosed cases of oral squamous

cell carcinoma, in Karpaga Vinayaga Institute of Medical Sciences and

Research, to help the clinical consultant to plan the targeted and adjunctive

therapy regimens accordingly.In this study we have proven consistent results

similar to the literature studies for considering e cadherin and vimentin for

studying the association of EMT phenomenon taking place in advanced tumors

favouring risk of invasion and distant metastasis.

“a study of expression of e cadherin and...

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