Correlation of E-cadherin expression with differentiation grade and ...
“A STUDY OF EXPRESSION OF E CADHERIN AND...
Transcript of “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
9 52 F Bm WD neg HS neg
10 60 F Bm WD neg HS neg
11 60 F Tongue MD pos LS LS
12 65 F Bm WD neg HS neg
13 65 F Tongue MD neg LS HS
14 44 M Bm WD neg HS neg
15 62 F Bm WD neg HS neg
16 51 M Tongue WD neg HS neg
17 55 M Bm WD neg HS neg
18 57 M Ton-base PD pos neg HS
19 65 F Tongue WD neg HS neg
20 49 F Tongue WD neg HS neg
21 55 M 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
28 40 F Tongue WD neg HS neg
29 75 F Tonsil MD neg neg LS
30 45 M Tongue WD neg HS neg
31 75 F Tongue MD pos LS HS
32 53 M Tongue PD pos neg HS
33 47 M Tongue WD neg HS neg
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
46 47 M Tongue 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
50 40 M Tongue WD neg HS neg
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
12. Then the slides were washed in 2 changes of TBS buffer for 5 mins
each.
13. Then incubation with novolink polymer for 30 mins
14. Then the slides were washed in 2 changes of TBS buffer for 5 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)
E-cadherin +ve E-cadherin -ve
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)
Vimentin +ve Vimentin -ve
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
Fishers Exact Test 0.5675
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
Fishers Exact Test 0.0002
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)
E-cadherin +ve E-cadherin -ve
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)
Vimentin +ve Vimentin -ve
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
Fishers Exact Test 0.1211
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)
E-cadherin +ve E-cadherin -ve
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
Fishers Exact Test 0.0039
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
Vimentin +ve Vimentin -ve
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
Vimentin +ve Vimentin -ve
Nu
mb
er
of
Pat
ien
ts
E-cadherin +ve E-cadherin -ve
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
Vimentin +ve Vimentin -ve
Nu
mb
er
of
Pat
ien
ts
E-cadherin +ve E-cadherin -ve
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.