Significance of HER-2/neu Overexpression in Oesophageal Carcinomas and It’s Correlation with the Histopathological Grading

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SIGNIFICANCE OF HER-2/NEU OVEREXPRESSION IN OESOPHAGEAL CARCINOMAS AND IT’S CORRELATION

WITH THE HISTOPATHOLOGICAL GRADING

Dissertation submitted in

Partial fulfillment of the regulations required for the award of M.D. DEGREE

In

PATHOLOGY – BRANCH III

THE TAMILNADU

DR. M.G.R. MEDICAL UNIVERSITY CHENNAI

APRIL 2016

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DECLARATION

I hereby declare that the dissertation entitled “SIGNIFICANCE

OF HER-2/NEU OVEREXPRESSION IN OESOPHAGEAL

CARCINOMAS AND IT’S CORRELATION WITH THE HISTOPATHOLOGICAL GRADING” is a bonafide research work done by me in the Department of Pathology, Coimbatore Medical College

during the period from July 2014 to July 2015 under the guidance and

supervision of Dr.A.ARJUNAN, M.D., Professor, Department of Pathology, Coimbatore Medical College.

This dissertation is submitted to The Tamilnadu Dr.MGR Medical

University, Chennai towards the partial fulfilment of the requirement for

the award of M.D., Degree (Branch III) in Pathology. I have not

submitted this dissertation on any previous occasion to any University for

the award of any Degree.

Place: Coimbatore

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CERTIFICATE

This is to certify that the dissertation entitled “SIGNIFICANCE

OF HER-2/NEU OVEREXPRESSION IN OESOPHAGEAL

CARCINOMAS AND IT’S CORRELATION WITH THE HISTOPATHOLOGICAL GRADING” is a record of bonafide work done by Dr.SARANYA.V in the Department of Pathology, Coimbatore Medical College, Coimbatore under the guidance and supervision of

Dr.A.ARJUNAN,M.D, Professor, Department of Pathology, Coimbatore Medical College and submitted in partial fulfilment of the requirements

for the award of M.D. Degree (Branch III) in Pathology by The

Tamilnadu Dr. MGR Medical University, Chennai.

Guide Head of the Department

Dr.A.ARJUNAN, M.D., Dr.C.LALITHA, M.D.,

Professor, Professor,

Department of Pathology, Department of Pathology, Coimbatore Medical College, Coimbatore Medical College,

Coimbatore. Coimbatore.

Dr.A.EDWIN JOE.M.D.B.L.

The Dean,

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ACKNOWLEDGEMENT

To begin with, I thank the Almighty in making this project a

successful one.

I express my deep gratitude to Dr.A.EDWIN JOE, M.D., B.L.,

Dean, Coimbatore Medical College, for permitting me to undertake this

study.

I express my sincere gratitude to Dr.C.LALITHA, M.D.,

Professor and Head, Department of Pathology, Coimbatore Medical

College, for having suggested this topic for dissertation and for having

rendered her valuable support and encouragement without which this

project work would not have been feasible.

I am extremely grateful to my guide Dr.A.ARJUNAN, M.D., Professor, Department of Pathology, Coimbatore medical college for his

valuable guidance, suggestions, constant encouragement and support

during this endurable work.

I also wish to record my sincere thanks to all Associate and

Assistant Professors of Department of Pathology, Coimbatore Medical

College, for their constant support and encouragement throughout the

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I also thank Dr.R.D.Puvitha,M.D., Assistant professor, Department

of Pathology for her valuable suggestions and guidance throughout my

study.

I thank all the technical staffs in the Department of Pathology,

Coimbatore Medical College, for their sincere and timely technical

assistance.

I express my heartfelt thanks to Department of Surgery and

Surgical Oncology, Coimbatore Medical College, for their constant

support throughout the course of this study.

It would not be complete without mention of my husband

Dr.Prakash.V, and my mother in law Mrs.Girija Venugopal for their

constant support ,encouraging words and source of strength all the way

through this endeavour.

To my lovable son P.S.Srirag , my parents and my friends,I

express my gratitude for the moral support and patience while pursuing

this study.

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CONTENTS

SI.NO. PARTICULARS PAGE NO.

1. INTRODUCTION 1

2. AIMS & OBJECTIVES 3

3. REVIEW OF LITERATURE 4

4. MATERIALS AND METHODS 50

5. OBSERVATION AND RESULTS 57

6. DISCUSSION 82

7. SUMMARY AND CONCLUSION 89

8. BIBLIOGRAPHY

9. ANNEXURES

ANNEXURE I - PROFORMA

ANNEXURE II – MASTER CHART

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LIST OF TABLES

SI.NO TITLE PAGE NO

1 Age wise distribution of oesophageal carcinomas 58

2 Sex wise distribution of oesophageal carcinomas 60

3 Location wise distribution of oesophageal

carcinomas

61

4 Tumor types 63

5 Distribution of histological grades of

oesophageal carcinomas

64

6 Frequency of Her-2/neu positivity in oesophageal

carcinoma

66

7 Association of HER-2/neu expression with age 68

8 Association of HER-2/neu expression with sex 70

9 Association of Her-2/neu expression with

location

72

10 Her-2/neu expression in histological grades of

SCC

74

11. Her-2/neu expression in histological grades of

Adenocarcinoma

76

12 Association of Her-2/neu expression with staging 78

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LIST OF CHARTS

SI.NO TITLE PAGE NO

1 Age wise distribution of oesophageal carcinomas 59

2 Sex wise distribution of oesophageal carcinomas 60

3 Location wise distribution of oesophageal

carcinomas

62

4 Tumor types 63

5 Distribution of histological grades of

oesophageal carcinomas

65

6 Frequency of Her-2/neu positivity in

oesophageal carcinoma

67

7 Association of HER-2/neu expression with age 69

8 Association of HER-2/neu expression with sex 71

9 Association of Her-2/neu expression with

location

73

10 Her-2/neu expression in histological grades of

SCC

75

11 Her-2/neu expression in histological grades of

Adenocarcinoma

77

11 Association of Her-2/neu expression with

staging

79

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LIST OF COLOUR PLATES

SI.NO TITLE

1 Well differentiated squamous cell carcinoma H&E(10X)

2 Moderately differentiated squamous cell carcinoma

H&E(10X)

3 Poorly differentiated squamous cell carcinoma H&E(10X)

4 HER-2/neu expression in grade I SCC (10X)

5 HER-2/neu expression in grade I SCC (40X)

6 HER-2/neu expression in grade II SCC (10X)

7 HER-2/neu expression in grade II SCC (40X)

8 HER-2/neu expression in grade III SCC (10X)

9 Well differentiated adenocarcinoma H&E (10X)

10 Moderately differentiated adenocarcinoma H&E (10X)

11 Poorly differentiated adenocarcinoma H&E (10X)

12 Poorly differentiated adenocarcinoma H&E (40X)

13 HER-2/neu expression in grade I ADC (10X)

14 HER-2/neu expression in grade I ADC (40X)

15 HER-2/neu expression in grade II ADC(10X)

16 HER-2/neu expression in grade II ADC (40X)

17 HER-2/neu expression in grade III ADC (10X)

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1

INTRODUCTION

Oesophageal carcinoma has been rated worldwide as the sixth most

common cause of mortality due to cancer. There are two common types.

They are squamous cell carcinoma and oesophageal adenocarcinoma.

However, it is oesophageal SCC that predominates globally over

adenocarcinomas but the frequency of occurrence of oesophageal

adenocarcinoma has dramatically increased during the last few decades.

Owing to the elasticity of the oesophagus and aggressiveness of the

tumor growth , oesophageal carcinomas usually proceed to the advanced

stage prior to the diagnosis. The mortality rate still remains high despite

the development of advanced therapeutic modalities apart from surgical

resection.

Hence, this study has been undertaken to assess the expression of

HER-2/neu receptor in the various degrees of differentiation especially

Squamous cell carcinomas and Adenocarcinomas and thereby any

positive result could be used for treating patients with anti-HER-2/neu

monoclonal antibodies (Herceptin) to improve the patient’s survival .

In this aspect, Amplification of human epidermal growth factor

receptor 2/neu (2/neu) gene and the overexpression of the

(15)

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various studies. The study is carried on with an idea that patients with

these cancers having dismal survival rates may be benefitted by the

identification of certain possible molecular targets such as HER-2/neu.

There are some reported cases of carcinomas which express

HER-2/ neu having poorer prognosis and fail to respond to the conventional

chemotherapy. Such tumors respond well to specific targeted therapy

with HER-2/neu antibody (Trastuzumab) thereby prolonging the survival

(16)
(17)

3

AIM OF THE STUDY

Aim is to correlate the immunohistochemical expression of HER-

2/neu with the histological grade of oesophageal carcinomas.

OBJECTIVES

 To study the immunohistochemical expression of HER-2/neu in

oesophageal carcinomas.

 To correlate the HER-2/neu expression with age, gender and the type

of the carcinoma.

 To study the correlation of HER-2/neu expression with

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REVIEW OF LITERATURE

EMBRYOLOGY AND DEVELOPMENT OF OESOPHAGUS:

The embryonic development of oesophagus begins as a bud on the

ventral aspect of the foregut between twenty third and twenty sixth day. It

grows downwards to become a diverticulum. As it grows caudally the

oesophagus gets separated posteriorly from tracheal lumen by the fusion

of two lateral folds towards the midline. The oesophagus elongates with

separation, mainly due to rapid increase in growth at cranial end.1 This

separation is finally accomplished by thirty five to forty days.

Initially the lining is stratified columnar, which later develops into

ciliated cells. These ciliated cells finally transform into stratified

squamous epithelium. This usually begins in the middle third of the

oesophagus. By term this squamous lining is complete. But small islands

of ciliated epithelium still exists in post natal life. By the eighth week of

gestation, the circular muscle coat appears, and by thirteenth week of

gestation, the longitudinal muscle layer develops. Ganglion cells and

(20)

5 ANATOMY OF OESOPHAGUS:

Oesophagus is a long fibromuscular conduit measuring about 25cm

in length.It lacks any secretory or absorptive function. It extends from the

pharynx at the level of the cricoid cartilage opposite to the sixth cervical

vertebra upto the cardia of the stomach , opposite to the tenth - eleventh

thoracic vertebra. For endoscopists, the length of the oesophagus is

measured from the incisor teeth. It pierces the left diaphragmatic crus and

has an intra abdominal portion for a length of 1.5cm.3

It has two sphincters. The upper oesophageal sphincter is 3cm

segment of proximal oesophagus at the level of cricopharyngeus muscle,

whereas the Lower oesophageal sphincter is about 2-4 cm segment just

above the gastro oesophageal junction at the level of the diaphragm.4

Oesophagus has three levels of constrictions. First constriction at

about 15cm from the upper incisors and is the narrowest portion. Second

constriction is at about 23cm from the upper incisors the point at which

the oesophagus is crossed over by the aortic arch. Third constriction is at

about 40cm from the upper incisor teeth the point where the oesophagus

(21)

6 RELATIONS OF THE OESOPHAGUS:

In point of view of the spread of the local carcinoma , it’s principal

relations that are of importance to a pathologist in assessing the local

spread of the tumour are- trachea, left main bronchus, aortic arch,

descending aorta an left atrium.

LYMPHATIC DRAINAGE:

Lymphatic channels drain the upper third of the oesophagus into

the deep cervical nodes via the paratracheal nodes.Lymphatic channels

drains the lower third of the oesophagus into posterior mediastinal nodes.

MUCOSAL DEFENCES:

Pre-epithelial, epithelial, and submucosal defenses play a very

important role in protecting the esophagus from injury. Pre-epithelial

defenses consist of the combined actions of the LES and the esophageal

muscles to reduce the reflux of gastric contents and promote clearance of

refluxed material. The micro ridges on squamous cells holds the mucus

on their surfaces, thus providing a protective coat. The esophageal

epithelium is also protected by a mucosal bicarbonate barrier and

hydrophobic surfactants which are secreted by submucosal glands.

(22)

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involve regulation of blood supply via responses of nerves, mast cells,

and the blood vessels.

HISTOLOGY OF OESOPHAGUS:

Oesophageal wall is composed of mucosa, submucosa, muscularis

propriae and adventitiae. The mucosa is composed of lining

epithelium(stratified non-keratinized squamous, basal layer comprising

1-4 cells thickness, scattered melanocytes and neuroendocrine

cells).Lamina propria(composed of loose connective tissue containing

few mucin secreting oesophageal cardiac glands) and muscularis mucosa

is thicker which can undergo re-duplication in Barrett Oesophagus,a

feature of importance in staging adenocarcinomas arising from this

background.

The submucosa contains mucous glands.The muscularis propria is

composed of smooth and skeletal muscle bundles in the upper one fourth

of the oesophagus and the remaining portions composed of only smooth

muscle seen as inner circular and outer longitudinal layer.

The oesophagus lacks serosal layer, except for its lowermost

portion.Very few meissner’s plexuses are found in submucosa and

(23)

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Identification of gastro-oesophageal junction is practically difficult

and ideally be at the proximal limit of histological gastric oxyntic

mucosa.10,11

CYTOLOGY OF THE NORMAL OESOPHAGUS

The exfoliated oesophageal cells from the mucosa includes the non

keratinized superficial squamous cells, intermediate cells, and, rarely,

parabasal cells. Epithelial pearls may occasionally be found whose

presence in large numbers suggest an inflammatory or erosive lesion.

Gastric-type columnar cells derived from the distal esophagus or from

islands of gastric mucosa associated with Barrett esophagus may be seen.

(24)

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Oesophagitis has numerous causes, the most common being

gastroesophageal reflux, infections, and drugs. It is of importance to

assess the consequences of the inflammation, to follow the course of the

underlying disease, and to treat the etiology. Irrespective of the cause,

most cases of oesophagitis share common histologic features.

Oesophageal inflammation might be acute, chronic, or mixed. Changes

associated with acute damage include ballooning of cells and presence of

inflammatory cells like eosinophils and neutrophils. Features such as

basal cell hyperplasia and papillary elongation develops. In severe

oesophagitis, ulcers, erosions, or neutrophils may be seen. Chronicity of

the epithelial damage leads to submucosal fibrosis or strictures. Patients

with longstanding reflux esophagitis are predisposed to develop Barrett

oesophagus .

The most common cause of esophageal inflammation is

gastroesophageal reflux disease. GERD-induced oesophagitis is a term

used for by reflux of gastric contents into the oesophagus, which causes

injury to the oesophageal mucosa. In the Western countries due to

change in the dietary lifestyle GERD has shown an alarming rise in

incidence .Clinically, it has been classified as erosive or nonerosive

(25)

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Risk factors associated with GERD include ageing and certain

lifestyle habits, such as consumption of alcohol and tobacco smoking.

GERD affects people of all ages, but recent statistics show that the risk

increases with age. The major event in GERD is contact and injury to the

epithelium lining the oesophagus by gastric acid due to persistant loss of

tone of lower oesophageal sphincter.13

Endoscopically it appears as irregular white hyperaemic patches /

plaques which are the earliest changes. Histologically, these white

patches correspond to acanthosis of the epithelium because of the

presence of clear squamous cells. These are PAS negative (different from

glycogenic acanthosis). Patients with reflux symptoms for a period of 5

years or longer should undergo endoscopy to screen for Barrett

oesophagus.14

BARRETT OESOPHAGUS:

Barrett oesophagus is the long term complication of Chronic

GERD that is characterized by intestinal metaplasia within the

oesophageal squamous mucosa. The greatest concern is the increased risk

of oesophageal adenocarcinoma. Genomic sequencing done on biopsies

from Barrett oesophagus have revealed the presence of mutations that are

(26)

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Chronic GERD leads to ulceration and inflammation of the normal

oesophageal squamous mucosal lining which, if persistent and recurrent,

ultimately leads to intestinal metaplasia(Barrett Oesophagus) which is

characterized by the presence of goblet cells. Theoretically, it could be

due to the migration of the columnar cells from the gastric mucosa, or

because of phenotypic switch in the undifferentiated elements in the stem

cell population/from underlying submucosal glands.

MORPHOLOGY:

Endoscopically, it can be visualized as one or several patches of

red,velvety mucosa extending upward from the GEJ. This metaplastic

mucosa is accompanied by residual smooth, pale squamous (oesophageal)

mucosa. The sensitivity of Barrett oesophagus has been increased by high

resolution endoscopes which has led to sub-classification of Barrett

oesophageal segment as long segment which involves 3cm or more and

short segment which involves less than 3cm.

Microscopically,the squamous oesophageal epithelium is replaced by intestinal-type metaplasia with goblet cells. These goblet cells are

diagnostic of Barrett oesophagus which have mucous vacuoles staining

(27)

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wine goblet to the cells. There may also be present the gastric type

foveolar cells which are the non-goblet cells.15

EPITHELIAL DYSPLASIA IN BARRETT OESOPHAGUS:

Dysplasia is defined as unequivocal neoplastic epithelium that

remains confined above the basement membrane of the epithelium from

which it developed and literally means disordered growth. Many

pathologists prefer the recently proposed Vienna Classification System

for grading of dysplasia in Barrett oesophagus.16

VIENNA CLASSIFICATION SYSTEM

Negative for neoplasia /dysplasia

Indefinite for neoplasia /dysplasia

Non invasive low grade dysplasia

Non invasive high grade dysplasia

Invasive neoplasia

Negative for dysplasia is typically applied to cases of Barrett Oesophagus that show metaplastic columnar epithelium without

dysplastic features. Dysplasia has to be distinguished from regenerative

epithelial changes. Features similar to both conditions are nuclear

enlargement and hyperchromasia, increased mitosis etc. But in

(28)

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evenly spaced, round – oval nuclei with smooth nuclear contours and

most importantly they have low nuclear to cytoplasmic ratio. Endoscopic

biopsies diagnosed as high grade dysplasia is often associated with

invasive carcinoma.

Indefinite for dysplasia refers to when regenerative changes particularly in areas of active inflammation or ulceration occur which

could not be distinguished from dysplasia histologically.

Positive for dysplasia: Oesophageal biopsies evaluated for dysplasia in Barrett oesophagus are classified low or high grade. Features

such as nuclear hyperchromasia, atypical mitosis, irregular clumped

chromatin, increased nuclear to cytoplasmic ratio are seen. There is

failure of maturation of the epithelial cells as they migrate to oesophageal

surface. Gland formation is frequently abnormal which is characterized

by budding, irregular shapes and cellular crowding. High grade dysplasia

exhibits both cytological and architectural changes.17

CARCINOMA IN BARRETT OESOPHAGUS:

The most important complication of Barrett esophagus is

carcinoma, which is nearly always accompanied and preceded by

dysplasia. This dysplasia noted in areas of intestinal metaplasia,should be

(29)

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inflammatory injury 18.Adenocarcinoma falls into majority of the invasive

carcinomas developing in Barrett oesophagus. It has been estimated that

Barrett-associated adenocarcinoma accounts for 5–10% of all esophageal

carcinomas.Most common molecular alterations known to be associated

in specimens of Barrett esophagus containing lesions within the dysplasia

- adenocarcinoma sequence are TP53 mutation and overexpression,

altered expression of various apoptosis-related proteins.19

PROGNOSIS:

Adenocarcinoma arising in the setting of Barrett oesophagus has

poor prognosis, with a 5-year survival rate of 14.5%. Stage by stage, this

prognosis seems to be similar to that of conventional squamous cell

carcinoma of the oesophagus.20

NATURAL HISTORY OF OESOPHAGEAL CARCINOMA

Oesophageal carcinomas is one of the most aggressive tumours.

Two histological types occur in the oesophagus SCC and ADC. SCC is

the most frequent histological type predominating in the developing

countries and ADC in the western areas. These tumours require high

clinical and surgical attention because of a very poor survival rate despite

(30)

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WHO CLASSIFICATION OF TUMOURS OF THE OESOPHAGUS

EPITHELIAL TUMOURS

Premalignant lesions

Squamous

Intraepithelial neoplasia, low grade

Intraepithelial neoplasia, high grade

Glandular

Dysplasia, low grade

Dysplasia, high grade

Carcinoma

Squamous cell carcinoma

Adenocarcinoma

Adenoid cystic carcinoma

Adenosquamous carcinoma

Basaloid squamous cell carcinoma

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Spindle cell carcinoma

Verrucous carcinoma

Undifferentiated carcinoma

Neuroendocrine neoplasms (carcinoid)

Neuroendocrine carcinoma

Large cell NEC

Small cell NEC

Mixed adeno neuroendocrine carcinoma

MESENCHYMAL TUMOURS

Granular cell tumour

Hemangioma

Leiomyoma

Lipoma

Gastrointestinal stromal tumour

Kaposi sarcoma

(32)

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Melanoma

Rhabdomyosarcoma

Synovial sarcoma

Lymphoma

Secondary tumours

DYSPLASIA IN SQUAMOUS EPITHELIUM

Squamous cell carcinoma of the oesophagus begins as an insitu

lesion termed as Squamous dysplasia. Dysplasia is defined as the

presence of unequivocal neoplastic cells which remains confined to the

epithelium. Squamous dysplasia is more common in patients who are at

high risk for squamous cell carcinoma and is adjacent to squamous cell

carcinomas in 60% to 90% of cases.

GROSS PATHOLOGY

Dysplastic epithelium appears friable, erythematous and irregular

in almost 80% of cases. The lesion may present as either an erosion,

plaque or nodule. However, dysplastic epithelium may appear completely

normal endoscopically. Dysplastic cells may also be retrieved by

exfoliative balloon cytology, a technique often used for screening in

(33)

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Low-grade dysplasia shows involvement of the basal third to half

of the squamous epithelium, whereas high-grade lesions show near

complete involvement of all the layers of the squamous epithelium.

Cytologic changes include pleomorphism, increased

nucleus-to-cytoplasm ratio, nuclear hyperchromasia, and increased mitotis. Nucleoli

may be present and are not specific because they are also frequently

present in reactive squamous epithelium.21

INVASIVE SQUAMOUS CELL CARCINOMA

Oesophageal squamous cell carcinoma is a malignant tumour of

epithelial origin. It is named so because of it’s squamous cell

differentiation, typically characterized by keratinocyte-like cells with

intercellular bridges and/or keratinization.

EPIDEMIOLOGY:

Squamous cell carcinomas of oesophagus are more common in

men aged above 50 years with a female: male ratio being 1:3. According

to Mimura et al, the mean age was found to be 64.5 years.22 It is relatively

common in China and other Oriental countries. In Western countries,

there has been an epidemiologic switch from esophageal squamous cell

carcinoma associated with tobacco and alcohol to Barrett-related

(34)

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highlighted in the Eastern countries and in many developing countries.

Iran, Central China, South Africa and Southern Brazil are some of the

regions showing marked incidence rates. However, the incidence of this

cancer is most unlikely before the age of 30 in both high and low risk

areas. In the United States, a recent study shows that the SCC is 2-3 times

commoner in blacks.23

AETIOLOGY:

1. Alcohol and Tobacco:

The consumption of tobacco and alcohol accounts for almost 90%

of the risk of SCC in the western countries. Regarding the tobacco use, a

moderate use during a long period poses a higher risk.24 Tobacco and

alcohol have synergistic effect for increased risk of oesophageal

carcinomas.

In Japanese alcoholics, a polymorphism in ALDH2 gene that

encodes for aldehyde dehydrogenase 2,has shown a promising

association with several cancers of the upper digestive tract, including

squamous cell carcinoma. This research study suggests role of

acetaldehyde, as one of the main carcinogenic metabolites of alcohol in

(35)

20 2. Nutrition :

In high-risk areas like China, a deficiency in certain trace elements

and the consumption of pickled or mouldy foods (which are potential

sources of nitrosamines) have been suggested. Fungal Contamination of

these foodstuffs and pickled vegetables with species of Fusarium, and

Aspergillus initiates the formation of nitrosamines, which are potent

oesophageal carcinogens.

3. Hot beverages:

The consumption of burning-hot beverages has been posed as the

most common risk factors worldwide because it causes thermal injury

that leads to chronic oesophagitis which is an important precancerous

lesion.26

4. HPV:

In about 20-40% of the cases of SCC in the high risk areas like

china, HPV DNA is identified to be the major causal factor. However it

is absent in the cancers arising in Western countries . Human papilloma

virus (HPV) plays an important role in the etiology of epithelial cancers

of the cervix, vulva, anus, penis, and oropharyngeal cavity. However,

despite numerous studies, the role of HPV in the etiology of ESCC

(36)

21

HPV in oesophageal tumors, others have found HPV in up to 75% of

cancers. The inconsistency of these results could be differences in the

study design, geographic variation, or lack of appropriate adjustment for

tobacco use and alcohol consumption.27

5. Nitrosamines :

Recently, nitrosamines found in fungus contaminated food were

confirmed as one of the most powerful and stable carcinogenic factors in

EC.

6. Genetic susceptibility:

Familial predisposition of oesophageal cancer is not well

established except that there is a strong association with focal Non

Epidermolytic Palmoplantar Keratoderma (NEPPK ortylosis) which is an

autosomal, dominantly inherited disorder involving the plantar and

palmer surfaces of the skin. Cases of oesophageal cancer in families

associated with keratosis palmaris and plantaris (tylosis) inherited as a

dominant characteristic have been reported . Of late, studies like linkage

analysis has mapped the gene locus responsible for tylosis oesophageal

cancer (TOC) to chromosome 17q25 and it has been proven that this

locus is significantly being deleted in sporadic squamous cell

(37)

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a tumour suppressor gene for oesophageal squamous cell carcinoma at

this locus.28,29

7. Other risk factors:

Other predisposing conditions: achalasia, corrosive strictures,

squamous cell carcinoma of other aerodigestive tract sites, diverticula,

Plummer –Vinson syndrome and coeliac disease.

LOCALIZATION:

Squamous cell carcinoma of the oesophagus is located

predominantly in the middle and the lower third of the oesophagus, upper

third accounting for only about 10-15% of the cases.

CLINICAL FEATURES:

Symptoms and signs :

Dysphagia is one of the most common symptom of oesophageal

carcinoma. Apart from this, patient may also present with weight loss,

retrosternal or epigastric pain and regurgitation caused by narrowing of

(38)

23 DIAGNOSIS:

1. ENDOSCOPY AND STAINING:

Superficial SCC is viewed only as a mild elevation or shallow

depression on the mucosal surface which is a less significant

morphological change when compared to advanced stage SCC. Grossly

seen as flat, polypoidal or ulcerative lesions. Macroscopic diagnosis can

be facilitated by application of lugol’s iodine or toluidine blue. The

principle behind the application of toluidine blue is that it stains both

RNA and DNA, thereby staining areas richer in nuclei than the normal

mucosa and the lugol’s iodine stains the glycogen and the unstained areas

can be judged as precancerous and cancer lesions.31

2. ENDOSCOPIC ULTRASONOGRAPHY:

Oesophageal carcinoma presents on endosonography as a

circumscribed or diffuse wall thickening with a predominantly echo-in

(39)

24 3. IMAGING:

Tumour growth is characterized as swelling of the oesophageal

wall, with or without direct invasion to surrounding organs.

4. GROSS EXAMINATION :

The lesions in the early SCC may be polypoidal, plaque like,

ulcerated or depressing occult lesions. With regards to advanced stage of

SCC, here is the macroscopic classification defined in the Japanese

society for oesophageal diseases.

Fungating lesions- are exophytic or polypoidal growths which protrude into and obstruct the lumen.

Ulcerative pattern- in which the tumour is intramural growth with central ulcer having elevated edges.

Infiltrative pattern -which is predominantly diffuse marked by intramural growth.

The last two categories may invade the surrounding structures

including respitratory tree, aorta, mediastinum and pericardium.

(40)

25 5. HISTOPATHOLOGY:

Most of the invasive SCC are moderate to well differentiated, well

differentiated forms producing more keratin and poorly differentiated

forms producing less keratin.

Well differentiated SCC show nests of tumour cells which are polygonal epithelioid cells with ample eosinophilic cytoplasm, easily

visualised intercellular bridges, and abundant keratinization (epithelial

pearls), with relatively few compact basaloid cells.

Moderately differentiated tumors account for approximately two thirds of squamous cell carcinomas. They contain a higher proportion of

primitive basaloid cells than well-differentiated tumors and they are

typically arranged in trabaculae and irregular nests with only focal

keratinization.

Poorly differentiated SCC show receding degree of differentiation showing very minimal or no evidence of keratinization, the

tumour grows in solid sheets or as single cells, and may contain large,

bizarre pleomorphic cells and show increased mitosis with atypical

(41)

26 SUPERFICIAL SCC

Superficial Squamous cell carcinomas are those which are confined

only to the mucosa and submucosa, irrespective of the nodal status.

Histologically, they consist of tumour cells arranged in nests. These nests

of neoplastic cells invade into the lamina propria or submucosa. The

invading fronts have irregular borders, the degree of tumor differentiation

is variable. It is important to diagnose superficial carcinomas because it is

a potentially curable and carry a more favorable prognosis than advanced

cancers, with an improved overall 5-year survival rates of 65% to

90%.(stern) About 5% of superficial carcinomas invading the lamina

propria present with lymph node metastases in about 5% of the cases,

whereas in carcinomas that invade the submucosa the risk of lymph node

metastasis is about 35%..34

TUMOUR SPREAD AND METASTASIS:

Direct spread

In early stages the tumour spreads within the wall and at the later

stages the growth invades all the layers to involve the trachea, bronchi,

the lung parenchyma and the superior or posterior mediastinum.35

In all resected specimens it is very essential to take transverse

(42)

27

resection is clear even though it may appear uninvolved grossly.

Downward extension of oesophageal carcinoma into the stomach is very

rare.

Metastasis:

In almost 50-80% of the cases, by the time the diagnosis of

symptomatic oesophageal carcinoma is made, patient presents with

metastasis. Adenocarcinomas seem to metastasize earlier, and more

frequently, than the Oesophageal SCC .Frequently metastasis to the

regional lymph nodes occurs most commonly due to the presence of two

sets of lymphatics. One draining the mucosa and the other draining the

submucosa. It occurs in about 5% of the carcinomas that are confined to

the mucosa , and 30% in carcinomas that invade the submucosa and over

80% in carcinomas invading the adjacent tissues.36

Nodes likely to be affected include paratracheal, parabronchial,

paraoesophageal, posterior mediastinal, coeliac and upper deep cervical

groups. Those carcinomas involving the upper third of the oesophagus

usually spread to the cervical and mediastinal lymph nodes. Those lesions

in the middle third metastasise to the mediastinal, upper gastric and

cervical lymph nodes. Finally, carcinomas of the lower third spreads to

(43)

28

With respect to hematogenous route of spread , the most common

sites being the liver and lung . Other sites are bones, adrenal gland and

brain. A study shows 40% patients with metastatic SCC have deposits in

the bone marrow.38

VARIANTS OF SCC:

1. Verrucous carcinoma

2. Basaloid carcinoma

3. Adenosquamous carcinoma

4. Sarcomatoid variant

VERRUCOUS CARCINOMA

It is a rare variant of SCC grossly appearing as an exophytic,

warty, cauliflower-like growth which occurs in any part of the

oesophagus. Histologically, diagnosis may be difficult because of

extremely well differentiated nature with acanthosis , hyperkeratosis and

papillomatosis . The diagnosis is made by swollen and voluminous rete

(44)

29 BASALOID CARCINOMA

Incidence is 11.3 % of SCC’s. It is a very rare variant of

oesophageal SCC. But this variant is quite distinct from all other variants

which appears to be histologically similar to basaloid SCC of the upper

aerodigestive tract. Occurrence of this type of SCC is often noted in

elderly males and at an advanced age.

Microscopically the tumour has a biphasic pattern. One is the

basaloid component in the form of anastomosing cords/trabeculae , solid

sheets and microcystic structures. Individual cells are closely packed with

hyperchromatic nuclei and basophilic scanty cytoplasm, with a usual

solid pattern of growth and there are foci of comedome necrosis. It is

characteristically associated with high mitotic index .The other is the

neoplastic squamous component and this may be either insitu or invasive.

It can be closely mistaken for adenoid cystic carcinoma of

oesophagus. This is important to differentiate because adenoid cystic

carcinoma has a less aggressive course. However, prognostically

basaloid SCC does not seem to differ much from the conventional SCC of

(45)

30 ADENOSQUAMOUS CARCINOMA

These are very rare and aggressive tumours in which

adenocarcinomatous and squamous carcinomatous components are

intermingled in varying proportions or have a fairly clear boundary.

Most cases are reported in a columnar-lined (Barrett) oesophagus.

Histologically this condition mimicks mucoepidermoid carcinoma but

features like frequent presence of keratinization in the squamous

component and the marked nuclear pleomorphism in adenosquamous

carcinoma enables easy differentiation .

SARCOMATOID VARIANT

This variant usually appears as bulky polypoid growths , the site of

occurrence being the lower oesophagus . The tumour cells consist of a

‘sarcomatous’ component and the spindle cells are arranged in interlacing

bundles with bizarre giant cells. Areas of osseous and cartilaginous

metaplasia may be seen and this is mingled with an epithelial component

of squamous or undifferentiated carcinoma. These two components may

be intermixed in equal proportions or the spindle cell component may be

predominating. The transition from typical squamous carcinoma to the

(46)

31 OTHER CARCINOMAS:

SMALL CELL (NEUROENDOCRINE) CARCINOMA:

Other synonyms for this tumour are as oatcell carcinoma,

argyrophil cell carcinoma, neuroendocrine carcinoma, small cell

undifferentiated carcinoma and anaplastic carcinoma. These tumours are

often large, protuberant lesions arising in the middle and lower thirds of

the oesophagus. Microscopy shows small uniform tumour cells arranged

in sheets, ribbons or anastomosing cords and the individual cells are

round to polygonal having scanty cytoplasm and small hyperchromatic

nuclei .Nucleoli are inconspicuous. Crush artefact of the tumour cells is

common, especially in biopsy material. Rosette formation may be

present. Squamous differentiation has been described in some of the

cases.

CHORIOCARCINOMA

A few cases of choriocarcinoma of the oesophagus have also been

reported. These are large, exophytic, fungating tumours with extensive

areas of haemorrhage and necrosis which have known to occur mostly in

the lower part of the oesophagus. Some have been admixed with

adenocarcinoma, and two of the tumours occurred in a columnar-lined

(47)

32

present on histological examination, and one tumour showed yolk-sac

differentiation.41

MOLECULAR ALTERATIONS IN SCC

At the molecular level, the most commonly associated alterations

are overexpression of cell cycle regulatory proteins. They are cyclin D1

and cyclin E, and loss or inactivation of tumor suppressor proteins such

as p53, Rb, and p16 in up to 80% of cases. A high level of expression of

the epidermal growth factor receptor (EGFR), is seen in 29% to 92% of

the cases in some studies. Paradoxically, some of these alterations, such

as inactivating mutations of p53, seems to occur earlier in the course of

the neoplastic progression and are frequently detectable in squamous

dysplastic precursor lesions as well.

PROGNOSTIC FACTORS:42

Oesophageal SCC has a poor prognosis and the 5-year survival

rates in registries are around 10%. Cure can be a ray of hope only for

superficial cancer. The survival varies, depends upon tumour stage at

diagnosis, treatment given, patient’s general health status, morphological

features and molecular pathogenesis of the tumour. Other prognostic

factors are;

(48)

33

2. Stage: In situ and (intra)mucosal carcinomas are nearly always curable, and the superficial carcinomas have a definite cure rate

than the deeper tumors.

3. Lymph node metastases: Naturally this parameter is included in the staging system. Among those cases with positive lymph nodes,

cases with two or more involved nodes do worse than those with a

single node involvement. Thus a thorough pathologic examination

is essential. Before we label an esophageal carcinoma as pN0 it

has been stated that it is essential to examine a minimum of 10

lymph nodes. The expression of Bcl2 in the primary tumor has

shown significant association with an increased likelihood of nodal

metastases.43,44,49

4. Tumor length: Data from the SEER program have indicated that tumor length is an independent predictor of mortality.45

5. Microscopic grade: The importance of this parameter is controversial but, on the whole, not substantiated. Some studies

have shown a more favorable prognosis for the better differentiated

tumors, but grade loses most of its significance in multivariate

analyses.

(49)

34

associated with a worse prognosis, whereas peritumoral fibrosis

and lymphocytic reaction to the tumor were associated with a better

prognosis; however, only the latter feature was shown to be

statistically significant. The prognostic value of lymph vessel

invasion has been confirmed by other studies. It has also been

claimed that tumor vascularity and tumor budding correlate with

prognosis. 46

7. Surgical margins: There is high probability of local recurrence with involvement of circumferential surgical margins.Hence it is of

utmost importance to thoroughly examine the resected margins.47

8. DNA ploidy and proliferation index: A correlation between aneuploidy and either time to recurrence or survival has been found

in several studies but not in others. AgNOR counts have been said

to correlate with probability of recurrence in superficial carcinomas

and with overall prognosis.48

9. Epidermal growth factor receptor (EGFR): Overexpression of EGFR has been found to correlate with the histological grade of the

tumour, lymph node status, and poor prognosis. Tumors with

higher expression in both EGFR and transforming growth factor

(50)

35

10.P53 : Patients whose esophageal cancers express p53 are said to have a poorer prognosis.51

ADENOCARCINOMA OF OESOPHAGUS:

A malignant epithelial tumour of the esophagus with glandular

differentiation arising predominantly from Barrett mucosa in the lower

third of the oesophagus.52

LOCALISATION:

Adenocarcinoma usually arise in the distal part of the oesophagus

and may spread into the gastric cardia,in which case it is difficult to

differentiate from carcinomas arising from gastric cardia.53

EPIDEMIOLOGY:

Because of the overlapping incidence patterns and anatomic

proximity, there is a hypothesis that the origins of oesophageal and

gastric cardia adenocarcinomas are similar to one another but they have

different risk factors and prognosis. The incidence of squamous cell

carcinoma of the esophagus is greadually receding in the United States;

however, the development of adenocarcinoma from Barrett oesophagus

is showing an alarming rise in it’s incidence54

(51)

36

According to a study, approximately 98% of the cases of

oesophageal and gastric cardia adenocarcinomas occurred in whites and

85% were males. The median age at diagnosis was 66 years55.

PREDISPOSING FACTORS:

1. Barrett esophagus and gastroesophageal reflux:

Both Barrett esophagus and gastroesophageal reflux are strongly

associated with EADC. EADC arises from Barrett esophagus, which is a

metaplastic transformation of the normal stratified non keratinising

epithelium. With an increase in the length of Barrett mucosa, there is an

associated increase in risk of adenocarcinoma. It was reported that there

is a 30-fold increased risk of EADC in patients suffering from Barrett

oesophagus, whereas those with reflux but without Barrett oesophagus

had only a moderate increase in risk (3.1 times) when compared with the

general population.55

2. Intestinal metaplasia:

It is the replacement of the normal squamous oesophageal

epithelium by intestinal type of epithelium with goblet cells as a part of

the healing process after repetitive injury to the oesophageal mucosa and

(52)

37

The length of the oesophageal segment with intestinal metaplasia,

and the presence of ulcerations and strictures have been implicated as

further risk factors for the development of oesophageal adenocarcinoma

from reflux disease.56

3. Nutritional imbalance:

Both under-nutrition and over-nutrition are considered as risk

factors in Oesophageal adenocarcinomas and so are associated with

socioeconomic status.

4. Tobacco:

Smoking has been identified as another major risk factor for

oesophageal adenocarcinoma and may account for as much as 40% of

cases.

5. Alcohol:

In contrast to squamous cell oesophageal carcinoma, there is no

strong association between alcohol consumption and adenocarcinoma of

the oesophagus.

6. Obesity:

Increased body mass index is also found to be associated with an

(53)

38 7. Chemical carcinogens:

Polycyclic aromatic hydrocarbons produced by the partial combustion

of organic materials have also been implicated.

8. Genetics:

CDX homeobox genes CDX1 and CDX2 play an important role in intestinal epithelial differentiation.CDX1 mRNA and CDX2 mRNA are

expressed in gastric intestinal metaplasia which holds similarity with

intestinal metaplasia in oesophagus.57

COX-2 expression of the proinflammatory enzyme Cyclooxynage-2 (COX-Cyclooxynage-2) in the oesophagus increases with exposure to acid. COX-Cyclooxynage-2

mRNA is elevated in reflux associated squamous mucosa of the

oesophagus rather than the mucosa not associated with reflux, and

expression in patients with Barrett mucosa was shown to increase with

time, which may be due to chronic and repeated exposure to

gastroesophageal reflux.58

EGFR:

Induction of CDX2 by deoxycholic acid occurs through ligand

dependent activation of the epidermal growth factor receptor

(54)

39

EAC and in 20% of Barrett mucosa but a later study found EGFR

amplification in only 8% of EAC. Using semiquantitative IHC, EGFR

was reported to be overexpressed in 35% of Barrett mucosae with

high-grade dysplasia (HGD) and 80% of EAC when compared to Barrett

mucosae alone.59

CLINICAL FEATURES:

Very often dysphagia is the presenting symptom of advanced

oesophageal adenocarcinoma. This may be associated with retrosternal

epigastric pain in early stages and cachexia at the later stages.

DIAGNOSTIC MODALITIES:

ENDOSCOPY:

In early stages of adenocarcinoma, the lesion endoscopically

appears as a small polypoidal lesion, often it may appear flat, elevated /

depressed. If no lesion is detected macroscopically, a systematic tissue

sampling is recommended, because high grade intraepithelial neoplasia

are often occult and multicenteric. But more commonly it appears as

stenosis in the lower third of the oesophagus axially with a polypoidal

(55)

40 ENDOSCOPIC ULTRASOUND :

There is display on the monitor, of certain specific echo patterns of

the mucosa and submucosa of the columnar lined oesophagus. This

procedure is however useful only for staging of the tumors that are

already diagnosed at endoscopy with biopsy. The tumor is always

hypoechoic .

GROSS EXAMINATION :

The site of occurrence of oesophageal adenocarcinoma is the distal

third of the oesophagus along with a segment of Barrett mucosa. This

Barrett mucosa is seen as salmon pink mucosa adjacent to the tumor.

In the earlier stages, the findings may be subtle with irregular

projections / plaques at the mucosal surface. But at the time of diagnosis

the tumor presents with infiltration of the wall of oesophagus. Advanced

lesions are often polypoidal / fungating. Rarely, a diffuse infiltrating

growth similar to linitis plastic of stomach can occur.60

HISTOPATHOLOGICAL EXAMINATION :

Adenocarcinomas are graded similar to ESCC as well, moderately,

or poorly differentiated. The AJCC grading system classifies tumors by

(56)

41

In well-differentiated carcinomas >95% of the tumor is composed of glands which are almost entirely cystic or glandular or

irregularly shaped tubules seen infiltrating the mucosa, submucosa, and

muscularis.

In moderately differentiated carcinomas 50% to 95% of tumor is composed of glands with tumor cells arranged in irregular clusters and

solid sheets. In these tumors, the tumor cells within the glandular profiles

show stratification and may attain a cribriform pattern .

Poorly differentiated carcinomas 5% to 49% of tumor is composed of glands often seen diffusely infiltrating the oesophageal wall

with a prominent desmoplastic stromal reaction. The tumor cells are

arranged in diffuse sheets and poorly formed glandular lumina. Signet

ring cells and bizarre pleomorphic tumor cells may be present. It can

spread distally to the stomach. Just like in SCC, there is extension into

adventitial tissue and nearby structures.

MORTALITY AND SURVIVAL:

There is no actual difference between men and women regarding

the survival rate. Incidence of SCC is more common in blacks, whereas

adenocarcinoma is more common in whites. Survival rate varies with

(57)

42

are 75% in superficial SCC.Following surgery with chemoradiotherapy,

33 to 50 % of patients are alive without recurrence for 2 years. However,

most patients still present with advanced disease at the time of diagnosis

and hence survival remains poor.61With respect to adenocarcinoma ,

because of advanced stage at the time of diagnosis ,the overall 5 year

survival is less than 25% in contrast to the 80% survival for those

adenocarcinomas which are confined to the mucosa and submucosa.

TNM STAGING SCHEME FOR OESOPHAGEAL CARCINOMA

Primary tumour

TX primary tumour cannot be assessed.

T0 No evidence of primary tumour

T1 tumour invades lamina propria, muscularis mucosae, or

Submucosa

T1a tumour invades lamina propria and muscularis mucosae

T1b tumour invades submucosa

T2 tumour invades muscularis propria

(58)

43

T4 tumour invades adjacent structures

T4a resectable tumour invading pleura, pericardium or

Diaphragm

T4b unresectable tumour invading other adjacent structures

such as aorta, vertebral body , trachea, etc.

Regional lymph nodes

NX regional lymph nodes cannot be assessed.

N0 no regional lymph node metastasis

N1 metastasis in 1-2 regional lymph nodes

N2 metastasis in 3-6 regional lymph nodes

N3 metastasis in seven or more regional lymph nodes

Distant metastasis (M)

M0 no distant metastasis

(59)

44 HISTOLOGICAL GRADE (G)

GRADE 1 Well differentiated Squamous cell carcinoma/ adenocarcinoma

GRADE 2 Moderately differentiated grade

GRADE 3 Poorly differentiated grade

GRADE 4 Undifferentiated

IMMUNOHISTOCHEMICAL ANALYSIS IN OESOPHAGEAL CARCINOMAS

Immunohistochemistry was first conceptualised by Dr. Albert

Coons in 1941. It is a novel method of detection of cell or tissue antigens

by allowing a specific antibody to interact with the antigen epitopes. To

visualize the antigen-antibody reaction, the most commonly used method

is that the antibody is conjugated to an enzyme like peroxidase which

catalyses a colour reaction .It is an effective way to detect the exact

expression of the protein by the tissues.

In this aspect, there are many proteins expressed during the process

of tumorigenesis in esophageal carcinomas. By immunohistochemistry,

squamous cell carcinoma cells are positive for broad spectrum

(60)

45

29% of cases, but the majority of cases are negative for both cytokeratins

7 and 20. 62

THE HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR

There are four members in the epidermal growth factor receptor

(EGFR) family. HER1 ( ErbB-1), HER2 (HER2/neu, ErbB-2), HER3

(ErbB-3), and HER4 (ErbB-4). These are transmembrane receptor

tyrosine kinases.

The human epidermal growth factor receptor 2 (Her2 or ErbB-2)

was first described in 1986 in gastric carcinomas. The HER-2 gene is a

proto-oncogene and is located at the long arm of human chromosome

17q2163

The main function of HER-2/neu is involved in signal transduction,

leading to cell growth and differentiation. EGFR receptor undergoes

dimerization following ligand binding which results in phosphorylation

of the tyrosine kinase, triggering a cascade of intracellular signals of

which the most important pathway is the activation of mitogen-activated

kinase MAPK pathways. There is no known ligand for HER2 hence often

referred to as the orphan receptor.64Importantly, just as in breast cancer,

HER2 overexpression plays an important factor as a prognostic factor in

(61)

46

Japanese study which showed that the 5-year survival rates is

11% for HER2-positive cases and 50% for HER2-negative gastric

cancer.52 In certain preclinical studies, patients treated with trastuzumab

was found to inhibit the growth of oesophageal cancer cell lines. Based

on the preclinical data in oesophageal cancer and clinical evidence in

breast cancer, early phase trials of trastuzumab in patients with metastatic

oesophageal carcinoma which overexpressed HER2 have shown that the

antibody therapy against HER-2/neu has ended up in improved clinical

outcomes for the patient .65

Other molecular factors regulating the pathogenesis of oesophageal

carcinoma are:

a) p53: Of late , p53 gene mutations most often point mutations have been reported in almost 50% of the human cancers and they

appear to occur at early stages of oesophageal SCC and is said to

correlate with the tumour progression .Dietary carcinogens such

as alcohol and tobacco are said to promote p53 mutation.66

b) p16: p16/INK4A is a tumour suppressor gene that is located on chromosome number 9p21 downregulates cell cycle pathway and

(62)

47

Loss of this gene is said to occur in the early stages of

oesophageal carcinogenesis.67

c) Cyclin D: Cyclin D1 regulates cell cycle progression at the G1-S checkpoint. It is said to be associated with squamous cell

carcinoma of the oesophagus and hence, it’s overexpression is a

frequent feature of oesophageal carcinogenesis.68

d) Wnt /b-catenin signaling pathway: It is said to play a very important role in the normal cell growth and stem cell

maintainance and any aberration in the pathway leads to

tumorigenesis.

e) Cadherins: E-cadherins are transmembrane proteins that mediate intercellular adhesions. E-cadherin is mainly found in the

epithelial cells and it’s altered expression plays a role in the

pathogenesis of oseophageal SCC.

f) MAPK signaling: Mitogen–activated protein kinases are important components in the cascade of events regulating normal

physiological processes in the normal tisues and upregulation

leading to tumorigenesis.69

(63)

48

regulation of cell differentiation, apoptosis and cell growth.

Resistance to TGF induced growth inhibition was noted in many

neoplastic cells.70,71

TREATMENT

Patients with oesophageal carcinomas limited to the mucosa or

submucosa have an 80% to 100% 5-year survival rate, compared with

10% to 20% for those patients with tumors that extend into or through the

muscularis72. Lymph node metastasis and a higher number of positive

lymph nodes are also associated with reduced survival.73

The majority of oesophageal adenocarcinomas show involvement

into or through the muscularis propria at the time of clinical presentation.

Advanced tumors may spread directly into the adjacent structures like the

mediastinum, aorta, or stomach.

Adenocarcinomas without metastases to distant sites are treated

with oesophagectomy alone by conventional approach. Neoadjuvant

chemotherapy or radiotherapy has been advocated as a means of

improving resectability and survival, but it has not yet shown a definite

(64)

49

Treatment for squamous cell carcinomas: radiation therapy for

upper two third carcinomas and esophagogastrectomy for lower one third

carcinomas.Recent approach is combined modalities such as preoperative

radiotherapy followed by surgery or combined chemotherapy and

(65)
(66)

50

MATERIALS AND METHODS

STUDY DESIGN

This is a prospective study carried over a period of fifteen months

dated from July 2014 to July 2015 in the department of pathology at

Coimbatore medical College and Hospital, Coimbatore.

CASE SELECTION

Thirty oesophagectomy specimens were received at department of

pathology Coimbatore medical college during the above said period were

appropriately fixed in 10% neutral buffered formalin, detailed and

processed for routine H&E staining.

INCLUSION CRITERIA

All well fixed resected specimens diagnosed as oesophageal

carcinoma.

EXCLUSION CRITERIA

Improperly fixed oesophagectomy specimens are excluded from

(67)

51

HAEMATOXYLIN AND EOSIN STAINING FOR ROUTINE

MICROSCOPY

All oesophagectomy specimens received at the department of

Pathology, Coimbatore Medical College were appropriately fixed well in

10% formalin fixative. The specimens were examined macroscopically,

representative tumour bits were taken and were paraffin embedded.

About 4-6 micron thickness were cut onto glass slides for routine

Haematoxylin and Eosin staining

REAGENTS

1. Erlich’sHaematoxylin solution

2. Eosin Y solution

3. 1% acid alcohol solution

PROCEDURE

1. Deparaffinise the sections.

2. Dip the sections in xylene – (10 minutes) 2 changes.

3. Sections are placed in isopropyl alcohol –(10 minutes) 2 changes

4. Wash the sections are taken to water.

5. Then staining the sections with Ehrlich’s Hematoxylin–

(68)

52

6. Wash the sections briefly under tap water again.

7. Sections are differentiated in 1% acid alcohol.

8. And the sections are kept for blueing in running tap water.

9. The slides are counterstained with 1% eosin solution for 1 minute.

10. Sections are dehyrdrated with alcohol.

11. Then cleared in xylene and mounted with DPX.

The routine Haematoxylin and Eosin stained sections are observed

under light microscope and the histopathological types and grades were

assessed.

IMMUNOHISTOCHEMISTRY

The technique used is a two step indirect technique based on the

antigen detection in the cells and the tissues. The two step process is as

follows:

1. The specific epitopes on the cells bind to the primary antibody.

2. The following step is a calorimetric reaction which detects the

antigen antibody binding.

REAGENTS USED IN IMMUNOHISTOCHEMISTRY

(69)

53

2. Power block- it is a very effective universal protein blocking agent

which consists of casein and property additives in PBS with 15mm

sodium azide.

3. Chromogen: DAB- 3 3’ diaminobenzidine.

4. Liquid DAB substrate buffer is composed of peroxide and

stabilizers

5. Superenhancer reagent

6. Poly HRP reagent.

7. Mayer’s Haematoxylin – used for counterstaining.

8. Buffer solutions

BUFFER SOLUTIONS

TRIS BUFFER(PH 7.6)

TRIS buffer salt: 0.605gm

Sodium chloride: 8gm

Distilled water : 1000 ml

(70)

54 CITRATE BUFFER (pH 6.0)

Trisodiumcitrate : 2.94gm

Distilled water : 1000ml

1N Hydrochloric acid: 5ml

TRIS EDTA (pH 9.0)

TRIS buffer salt : 6.05 gm

Disodium EDTA : 0.74gm

Distilled water : 1000ml

PROCEDURE FOR IMMUNOHISTOCHEMISTRY

1. Sections are deparaffinised in xylene for thirty minutes.

2. Sections are then washed in absolute alcohol for five minutes

with two changes followed by tap water for ten minutes.

3. Sections are rinsed in distilled water for five minutes.

4. Next is the antigen retrieval which is accomplished by immersing

the slides in citrate buffer solution. This is followed by

microwaving at 450 degree celsius for ten minutes and at 800

(71)

55

5. Microwave step is followed by cooling to room temperature and

then slides are washed in distilled water.

6. Slides are now washed in TBS buffer for five minutes with two

changes. Sections are then subjected to treatment with peroxide

block for five to eight minutes followed by buffer wash for five

minutes with two changes.

7. Treat the slides with power block for ten minutes.

8. Then the slides are drained and the sections are covered with

primary antibody (supplied by Biogenex) for one hour.

9. This is followed by TBS buffer wash for five minutes with two

changes. Cover the sections with superenhancer for 30 minutes.

10. Sections are washed in TBS buffer for five minutes with two

changes. Cover the sections with poly HRP reagent for thirty

minutes.

11. The sections are again washed with TBS buffer for five minutes

with two changes.

12. Sections are then treated with DAB chromogen with substrate

Figure

TABLE 1 AGE WISE DISTRIBUTION OF OESOPHAGEAL CARCINOMAS

TABLE 1

AGE WISE DISTRIBUTION OF OESOPHAGEAL CARCINOMAS p.75
TABLE 2 SEX WISE DISTRIBUTION OF OESOPHAGEAL CARCINOMAS

TABLE 2

SEX WISE DISTRIBUTION OF OESOPHAGEAL CARCINOMAS p.77
TABLE 3 LOCATION WISE DISTRIBUTION OF OESOPAGEAL

TABLE 3

LOCATION WISE DISTRIBUTION OF OESOPAGEAL p.78
TABLE 4 TUMOUR TYPES

TABLE 4

TUMOUR TYPES p.80
TABLE 5 DISTRIBUTION OF THE HISTOLOGICAL GRADES

TABLE 5

DISTRIBUTION OF THE HISTOLOGICAL GRADES p.81
TABLE 6 FREQUENCY OF HER2 neu EXPRESSION

TABLE 6

FREQUENCY OF HER2 neu EXPRESSION p.83
TABLE 7 ASSOCIATION OF HER- 2/ NEU EXPRESSION WITH AGE

TABLE 7

ASSOCIATION OF HER- 2/ NEU EXPRESSION WITH AGE p.85
TABLE 9 ASSOCIATION OF HER-2 /NEU EXPRESSION

TABLE 9

ASSOCIATION OF HER-2 /NEU EXPRESSION p.89
TABLE 10 ASSOCIATION OF HER-2 /NEU WITH SCC HISTOLOGICAL

TABLE 10

ASSOCIATION OF HER-2 /NEU WITH SCC HISTOLOGICAL p.91
TABLE 11 ASSOCIATION OF HER-2 /NEU WITH ADENOCARCINOMA

TABLE 11

ASSOCIATION OF HER-2 /NEU WITH ADENOCARCINOMA p.93
TABLE 12 ASSOCIATION OF HER-2/NEU EXPRESSION WITH

TABLE 12

ASSOCIATION OF HER-2/NEU EXPRESSION WITH p.95
Fig 2. Moderately differentiated Squamous cell carcinoma  H&E (10x)

Fig 2.

Moderately differentiated Squamous cell carcinoma H&E (10x) p.100
Fig 1. Well differentiated Squamous cell carcinoma H&E (10x)

Fig 1.

Well differentiated Squamous cell carcinoma H&E (10x) p.100
Fig 3.Poorly differentiated  Squamous cell  carcinoma  H&E(10x)

Fig 3.Poorly

differentiated Squamous cell carcinoma H&E(10x) p.101
Fig 4. Weak focal 1+ membrane positivity of HER-2/neu in  grade I SCC (10x)

Fig 4.

Weak focal 1+ membrane positivity of HER-2/neu in grade I SCC (10x) p.102
Fig 5. Weak focal 1+ membrane positivity of HER-2/neu in  grade I SCC (40x)

Fig 5.

Weak focal 1+ membrane positivity of HER-2/neu in grade I SCC (40x) p.102
Fig 7. Moderate diffuse 2+ HER-2/neu positivity in  grade II SCC (40X)

Fig 7.

Moderate diffuse 2+ HER-2/neu positivity in grade II SCC (40X) p.103
Fig 6. Moderate diffuse 2+ HER-2/neu positivity in  grade II SCC (10X)

Fig 6.

Moderate diffuse 2+ HER-2/neu positivity in grade II SCC (10X) p.103
Fig 8. Strong 3+ membranous staining in grade III SCC (40x)

Fig 8.

Strong 3+ membranous staining in grade III SCC (40x) p.104
Fig 10. Moderately differentiated adenocarcinoma H&E (10X)

Fig 10.

Moderately differentiated adenocarcinoma H&E (10X) p.105
Fig 9. Well differentiated adenocarcinoma H&E (10X)

Fig 9.

Well differentiated adenocarcinoma H&E (10X) p.105
Fig 12.Poorly differentiated  Adenocarcinoma H&E (40X)

Fig 12.Poorly

differentiated Adenocarcinoma H&E (40X) p.106
Fig 11. Poorly differentiated adenocarcinoma H&E (10X)

Fig 11.

Poorly differentiated adenocarcinoma H&E (10X) p.106
Fig 13. Weak focal 1+ HER-2/neu positivity in grade  I Adenocarcinoma (10X)

Fig 13.

Weak focal 1+ HER-2/neu positivity in grade I Adenocarcinoma (10X) p.107
Fig 14. Weak focal 1+ HER-2/neu positivity in grade I Adenocarcinoma (40X)

Fig 14.

Weak focal 1+ HER-2/neu positivity in grade I Adenocarcinoma (40X) p.107
Fig 16. Moderate diffuse 2+ membranous positivity of HER-2/neu in grade II adenocarcinoma (40x)

Fig 16.

Moderate diffuse 2+ membranous positivity of HER-2/neu in grade II adenocarcinoma (40x) p.108
Fig 15.Moderate diffuse 2+ membranous positivity of HER-2/neu in grade II adenocarcinoma (10x)

Fig 15.Moderate

diffuse 2+ membranous positivity of HER-2/neu in grade II adenocarcinoma (10x) p.108
Fig 17.Diffuse strong 3+ HER-2/neu staining in grade III Adenocarcinoma  (10x)

Fig 17.Diffuse

strong 3+ HER-2/neu staining in grade III Adenocarcinoma (10x) p.109
Fig 18.Diffuse strong 3+ HER-2/neu staining in grade III Adenocarcinoma  (40x)

Fig 18.Diffuse

strong 3+ HER-2/neu staining in grade III Adenocarcinoma (40x) p.109

References