AN IMMUNOHISTOCHEMISTRY STUDY OF Ki67 AND p63

EXPRESSION OF SALIVARY GLAND NEOPLASMS IN A TERTIARY

CARE HOSPITAL OF SOUTHERN TAMILNADU

DISSERTATION SUBMITTED TO

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

CHENNAI

in partial fulfilment of

the requirements for the degree of

M.D. (PATHOLOGY)

BRANCH – III

TIRUNELVELI MEDICAL COLLEGE

TIRUNELVELI

APRIL-2017

CERTIFICATE

This is to certify that this Dissertation entitled “AN

IMMUNOHISTOCHEMISTRY STUDY OF Ki67 AND p63 EXPRESSION

OF SALIVARY GLAND NEOPLASMS IN A TERTIARY CARE

HOSPITAL OF SOUTHERN TAMILNADU” is the bonafide original work of

Dr.C.ARUNA MUTHARASI, during the period of her post graduate study from

2014 –2017, under my guidance and supervision, in the Department of

PathologyTirunelveli Medical College & Hospital, Tirunelveli, in partial

fulfillment of the requirement for M.D., (Branch III) in Pathology examination of

the Tamilnadu Dr.M.G.R Medical University will be held in April 2017.

Dr.K.SITHY ATHIYA MUNAVARAH, M.DThe DEAN

Tirunelveli Medical College,Tirunelveli - 627011.

CERTIFICATE

This is to certify that this Dissertation entitled “AN

IMMUNOHISTOCHEMISTRY STUDY OF Ki67 AND p63 EXPRESSION

OF SALIVARY GLAND NEOPLASMS IN A TERTIARY CARE

HOSPITAL OF SOUTHERN TAMILNADU” is the bonafide original work of

Dr.C.ARUNA MUTHARASI, during the period of her post graduate study from

2014 –2017, under my guidance and supervision, in the Department of

PathologyTirunelveli Medical College & Hospital, Tirunelveli, in partial

fulfillment of the requirement for M.D., (Branch III) in Pathology examination of

the Tamilnadu Dr.M.G.R Medical University will be held in April 2017.

Dr.K.Shantaraman,M.D Dr.K.Swaminathan,M.D

Professor and HOD of Pathology, Professor of Pathology,

Department of Pathology Tirunelveli Medical college

Tirunelveli Medical college Tirunelveli -11.

Tirunelveli -11.

DECLARATION

I solemnly declare that this dissertation titled “AN

IMMUNOHISTOCHEMISTRY STUDY OF Ki67 AND p63 EXPRESSION

OF SALIVARY GLAND NEOPLASMS IN A TERTIARY CARE

HOSPITAL OF SOUTHERN TAMILNADU” submitted by me for the degree

of M.D, is the record work carried out by me during the period of 2014-2016

under the guidance of Prof. Dr.S.Vallimanalan, M.D, and

Prof.Dr.K.Swaminathan, M.D, Professors of Pathology, Department of

Pathology, Tirunelveli Medical College, Tirunelveli. The dissertation is

submitted to The Tamilnadu Dr. M.G.R. Medical University, Chennai, towards

the partial fulfilment of requirements for the award of M.D. Degree (Branch

III)Pathology examination to be held in April 2017.

Place: Tirunelveli DR.C.ARUNA MUTHARASI,

Date: Post Graduate

Department of Pathology,

Tirunelveli Medical College,

Tirunelveli-11

ACKNOWLEDGEMENT

I take immense pleasure at this opportunity to acknowledge all those who

have helped me to make this dissertation possible. I express my heartfelt thanks

to the Dean, Tirunelveli Medical College, for permitting me to undertake this

study. I express my profound sense of gratitude to Dr.K.Shantaraman, MD.,

respected Professor and Head of Department of Pathology, Tirunelveli

MedicalCollege, Tirunelveli, for his valuable advice, constant guidance and

motivation in the preparation of this work.

I consider it my privilege and honour to have worked under the unstinted

encouragement, and supervision of Dr.S.Vallimanalan, M.D, and

DR.K.Swaminathan,M.D, Professors of Pathology.

I thank Dr.J.SureshDurai, MD., Dr.Arasi Rajesh, MD., and

Dr.Vasuki, MD., Professors of Pathology, for their constant support. I also thank

the Assistant Professors, for their encouragement. I take this

opportunity to thank all my postgraduate colleagues and all the technicians and

other members of the Department of Pathology for their constant help and

support throughout the tenure of this work.

I thank GOD ALMIGHTY, MY PARENTS and MY HUSBAND for

their blessings and support not only in this study but in all endeavours of my

life.

C.ARUNAMUTHARASI

ABBREVIATIONS

1. CN - Cranial Nerve

2. RER - Rough Endoplasmic Reticulam

3. H&E -Hematoxylin and Eosin

4. Ig - Immunoglobulin

5. EBV - Ebstein Barr Virus

6. HIV - Human Immunodeficiency Virus

7. EMA - Epithelial Membrane Antigen

8. CEA - Carcino Embryonic Antigen

9. CK - Cytokeratin

10.GFAP - Glial Fibrillary Acidic Protein

11.PTAH - PhosphoTungstic Acid- Hematoxylin

12.SMA - Smooth Muscle Actin

13.IHC - Immunohistochemistry

14.PAS - Periodic Acid – Schiff

15.PLGA - Polymorphous Low Grade Adenocarcinoma

16.CD - Cluster of Differentiation

17.DAB - Diamino Benzidine

18.HRP - Horseradish Peroxidase

19.TBS - Tris- Buffer Saline

20.Her 2/ neu - Human Epidermal Growth Factor -2

TABLE OF CONTENTS

S.NO TITLES PAGE NO

1 INTRODUCTION 1

2 AIMS AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4

4 MATERIALS AND METHODS 53

5 OBSERVATION AND RESULTS 58

6 DISCUSSION 70

7 SUMMARY 75

8 CONCLUSION 76

9 BIBLIOGRAPHY

10 ANNEXURES

1

INTRODUCTION

Salivary glands are one of the important exocrine glands of the body.

They constitute three pairs of major glands (Parotid, submandibular and

sublingual) and numerous minor salivary glands in the tongue, palate , cheek

and the lips. Saliva secreted by these glands help in keeping the oral cavity moist

and also aids in functions like chewing and swallowing. The salivary secretion

may be serous (parotid), mucus (sublingual) or mixed (submandibular glands).

Salivary gland neoplasms constitute about 0.4 to 13.5 cases per one lakh

population(1). The malignant neoplasms constitute 6% of all head and cancers

and 0.3% of all cancers(2). A variety of benign and malignant neoplasms arise

from the salivary glands.

A study for the incidence of salivary gland neoplasms showed that the

most frequent neoplasm of salivary gland is benign mixed tumour constituting

65.6% of salivary tumors, followed by Warthins forming 29.2% . Of the

malignant tumours, mucoepidermoid carcinoma is the commonest malignant

tumour constituting about 51.3% of all malignant tumours. The most common

site of involvement being Parotid (86%) followed by submandibular(14%)(3).

With the worldwide increase in incidence of salivary gland neoplasms ,

and with improvement in treatment quality of these neoplasms, it is essential to

properly identify, type and grade these tumours to aid in the early treatment of

the patients and thereby to improve survival and outcome.

Immunohistochemistry plays a vital role in assessing the diagnosis, grading

2

and prognosis of such tumours, which in turn aids in early and appropriate

treatment of these tumours.

3

AIMS AND OBJECTIVES

1) To study the pattern of expression of Ki 67 and p63 in various salivary

gland tumours.

2) To assess the significance of the expression of these markers in salivary

gland neoplasms.

4

REVIEW OF LITERATURE

SALIVARY GLAND

Salivary glands are one of the important exocrine organs of the body that

constitutes of major and minor salivary glands. The secretion of the salivary

gland is called saliva. It can be serous, mucus or mixed depending upon its

composition and properties(4). These secretions help in lubrication of oral cavity,

mastication, speech and deglutition. Saliva also has enzymes that aid in the

digestion of starch.

DEVELOPMENT OF SALIVARY GLANDS

The salivary glands develop from the epithelium of primitive oral cavity.

It develops as a thickening that later develops into the first branchial arch

(mandibular arch) mesenchyme that later forms a solid epithelial placode(5).

The placode protrudes into the mesenchyme and forms a solid mass of

cells. These cells are connected by means of a stalk to the tongue epithelium.

This stalk is composed of immature duct epithelial cells. It is followed by

development of small cleft like indendations on the surface of the epithelial

bud. Later, the primary bud is divided by clefts into multiple buds, and the

epithelium proliferates. The base of the cleft is formed by the primitive ductal

structure. Over the following days the primary bud divides multiple times. At

this stage the salivary gland undergoes repeated branching and finally the main

duct forms a lumen. Acini, which are the main secretory units of the salivary

5

gland, are formed by the end buds. By the time the lumen is completely formed,

the gland is composed of a network of ducts connecting the acini and the oral

cavity.

Salivary glands maturation and cellular differentiation occurs after

birth along with branching of salivary glands(6).

Figure 1: Development of salivary glands

6

MACROSCOPIC APPEARENCE

Parotid is the largest salivary gland. It contributes to 20% of salivary

secretions in an unstimulated gland which raises to 50% after stimulation(8). It

has a superficial and a deep lobe. The facial nerve, retromandibular vein and the

external carotid artery are enclosed by the Parotid. The gland is situated in the

pre - auricular area. It is paired and bilateral and is situated on either side upon

the mandibular ramus. It is encapsulated by the masseteric fascia. The parotid

plexus is formed by the branches of the facial nerve. It passes through the

parotid gland and divides it into a superficial and a deep part but does not

innervate it. The parotid duct is called as Stensen's duct. It opens through

parotid papilla opposite to the upper second molar tooth(8). Accessory parotid

glands are the minor glandular tissues situated along the parotid duct.

The masseter muscle is situated anterior to the partotid, and is bound

superiorly by the external acoustic meatus and the condyle of the mandible in the

glenoid fossa . The medial side is not covered by the capsule, the styloid

process and the transverse process of the atlas are visible on this side.

The parotid is supplied by the terminal branches of the external carotid

artery- maxillary arteries and superficial temporal arteries. The venous drainage

is by the retromandibular (or posterior facial) vein(9).

Parotid gland is innervated by many nerves from different sources. The

sensory supply is by the auriculotemporal nerve. The parasympathetic

7

innervation is mandibular branch of trigeminal nerve attained from the

glossopharyngeal nerve. Parasympathetic postganglionic neurons arising from

the otic ganglion, reach the gland via the auriculotemporal nerve.

Parasympathetic stimulation results in salivary secretion. Furthermore the gland

receives sympathetic innervation from direct fibers of the external carotid plexus.

Figure 2: Parotid gland with facial nerve

SUBMANDIBULAR SALIVARY GLAND.

It consists of a larger superficial part and a deep part(10). It contributes

to 65% of salivary secretion(8).

Superficial part - It lies at the angle of the jaw, situated between the

mandible and the mylohyoid muscle and overlapping the digastric muscle.

Posteriorly it lies close to the parotid gland, separated from it by the

stylomandibular ligament which is a condensation of its fascial sheath .

8

Superficially, it is covered by investing layer of deep cervical fascia just

beneath the platysma, It is crossed by the cervical branch of the facial nerve

(CN VII) and by the facial vein(11).

Deep part(smaller part)

It is related above to the mylohyoid muscle, medially to the hyoglossus

and styloglossus muscles, laterally to the mylohyoid muscle, superiorly to the

lingual nerve(CN V) and inferiorly to the Hypoglossal nerve (CN XII)(12).

The submandibular duct (Whartons duct ) originates from the deep part

of submandibular gland. It is about 5cm in length and exhibits a triple

relationship with the lingual nerve(13). It lies inferior to the ligual nerve at the

point of its exit from the gland, more distally the lingual nerve passes below the

duct and crosses the duct from the lateral to the medial side. The sublingual

gland lies immediately lateral to the submandibular duct. The duct opens at the

sublingual papilla or caruncle by the side of frenulum of tongue in the floor of

mouth(14).

The blood supply is derived from the branches of the facial and lingual

arteries. Venous drainage is carried out by the common facial vein and the

lingual vein.

Lymphatic drainage is by the submandibular lymph nodes that lies

between the gland and the fascial capsule. Occasionally some of these nodes may

be embedded deep within the gland.

9

SUBLINGUAL SALIVARY GLAND

The sublingual glands are major salivary glands and are the smallest and

most deeply situated. They contribute to only 7-8% of salivary secretion(8),

producing mixed secretions which are predominately mucous in nature. They are

situated under the tongue. They are laterally bordered by the mandible and

medially by genioglossus muscle.

On the anterior aspect, the glands of both sides unite and form a

horseshoe shaped structure around the lingual frenulum. The superior aspect of

this U-shape forms the sublingual fold, which is an elevated crest of mucous

membrane. Each sublingual fold travels anteriorly and joins the sublingual

papillae at the midline , on either sides of the lingual frenulum .

Minor sublingual duct ( duct of Rivinus), drain the sublingual glands.

There are 8-20 excretory ducts per gland, each opening out onto the sublingual

folds(15). In some people, a major sublingual duct ( duct of Bartholin) can be

present(16) . The sublingual duct then opens into submandibular duct through the

sublingual papillae.

Blood supply is via the sublingual and submental arteries which are

branches of the lingual and facial arteries respectively which are in turn

branches of external carotid artery. Venous drainage is by the sublingual and

submental veins, which are drained by the lingual and facial veins respectively,

which in turn drains into the internal jugular vein.

10

The sublingual glands are innervated by parasympathetic and

sympathetic fibres which regulate salivary secretions . Their innervation is

similar to that of the submandibular glands.

Figure 3: Macroscopic appearance of salivary glands

MICROSCOPIC APPEARANCE

SECRETORY PORTION

The secretory portions of salivary glands are composed of serous or

mucous or mixed secretory cells arranged in acini (alveoli) or tubules that are

surrounded by myoepithelial cells.

11

The secretory portions are formed by three types of cells

Serous cells are seromucous cells because they secrete both proteins that

give the serous quality and polysaccharides that give the mucoid

nature(17). These cells are pyramid shaped and have single, round, basally

located nuclei , a well-developed rough endoplasmic reticulum (RER)

and Golgi complex. They also have numerous basal mitochondria, and

abundant apically situated secretory granules which are rich in ptyalin –

a salivary amylase(18). They secrete products like kallikrein, lactoferrin,

and lysozyme(19). The basal regions of the lateral cell membranes form

tight junctions with each other. In front of the tight junctions,

intercellular canaliculi communicate with the lumen. The plasmalemma

basal to the tight junctions forms many processes that interdigitate with

those of neighbouring cells.

Mucous cells are similar in shape to the serous cells. Their nuclei are also

basally located but are flattened instead of being round like the serous

cell. In comparison to the serous cells, the mucous secretory cells have

fewer mitochondria, lesser RER, and a considerably greater amount of

Golgi apparatus, indicative of the greater carbohydrate component of their

secretory product . The apical region of the cytoplasm is occupied by

abundant secretory granules. The intercellular canaliculi and processes of

the basal cell membranes are much less extensive than those of serous

cells.

12

Myoepithelial cells (basket cells) share the basal laminae of the acinar

cells. They have a cell body that houses the nucleus and several long

processes that envelop the secretory acinus and intercalated ducts . The

cell body of the myoepithelial cells contains a small complement of

organelles in addition to the nucleus . It makes hemidesmosomal

attachments with the basal lamina(20). The cytoplasmic processes, which

form desmosomal contacts with the acinar and duct cells are rich in actin

and myosin. As the processes of myoepithelial cells contract, they press

on the acinus, facilitating release of the secretory product into the duct of

the gland.

Figure 4: Submandibular salivary gland with serous and mucinous

acini,H&E,40x

13

Figure 5: Cut section of salivary glands

DUCTAL PORTION

The ducts of the major salivary glands are branched structures. They

include the intercalated ducts, striated ducts, intralobular ducts, interlobular

ducts, interlobar ducts and finally the terminal ducts.

Intercalated ducts are the smallest branches of the system. The secretory

acini are attached to the intercalated ducts. The intercalated ducts are lined by a

single layer of small cuboidal cells and also have a myoepithelial layer lining.

Many intercalated ducts join to form the striated ducts .They are lined by

a single layer of cuboidal to low columnar cells(21). The basolateral membranes

14

of these cells are highly folded thereby dividing the cytoplasm into

compartments. These compartments contain elongated mitochondria thereby

giving a striated appearance to these cells. The basolateral cell membranes of

these cells have sodium adenosine triphosphatase . The sodium adenosine

triphosphatase pumps sodium out of the cell into the connective tissue, thus

conserving these ions thereby reducing the tonicity of saliva.

Striated ducts join with each other, forming intralobular ducts of

increasing caliber, which are surrounded by more abundant connective tissue

elements. The interlobular ducts are formed by the union of ducts draining the

lobules. The intralobar and interlobar ducts are in turn formed from the

interlobular ducts. The terminal duct of the gland delivers saliva into the oral

cavity.

SALIVARY SECRETION

The salivary gland initially produces primary saliva that is converted into

secondary saliva.

The major salivary glands produce around 1100mL of saliva per day(22).

Minor salivary glands located in the mucosa and submucosa of the oral cavity

contribute only to 5% of the total daily salivary output. To perform at this level,

salivary glands have an extraordinarily rich vascular supply of about 1ml per

gram of salivary gland per minute. During maximal secretion, the blood flow is

correspondingly increased.

15

Saliva performs innumerable functions like lubricating and cleansing of

the oral cavity, antibacterial activity, participating in the taste sensation by

dissolving food material, initial digestion via the action of ptyalin (salivary

amylase) and salivary lipase, aiding swallowing by moistening the food and

permitting the formation of bolus, and participating in the clotting process and

wound healing because of the clotting factors and epidermal growth factor

present in saliva.

The saliva produced by the acinar cells, called primary saliva, is isotonic

with plasma. The primary saliva is then modified by the cells of the striated

ducts by removing sodium and chloride ions from it and secreting potassium and

bicarbonate ions into it. This altered secretion, called secondary saliva, is

hypotonic(24).

Acinar cells and duct cells also synthesize the secretory component

required to transfer IgA from the connective tissue into the lumen of the

secretory acinus . Secretory IgA neutralises antigens in the saliva, thereby

protecting from their deleterious effects. Saliva contains lactoferrin, lysozyme,

and thiocyanate ion. Lactoferrin binds iron, an element essential for bacterial

metabolism: Lysozyme breaks down bacterial capsules, permitting the entry of

thiocyanate ions, a bactericidal agent, into the bacteria.

Salivary glands also secrete the enzyme Kallikrein into the connective

tissue. Kallikrein enters the bloodstream, where it converts kininogens, a family

16

of plasma proteins, into bradykinin, a vasodilator that dilates blood vessels and

enhances blood flow to the region.

LESIONS OF THE SALIVARY GLAND

EPIDEMIOLOGY

Salivary gland lesions are seen worldwide with increasing incidence rates.

The incidence of salivary gland lesions vary in different parts of the world. A 10

year study (2002 to 2011), conducted in NorthEastern India, shows that the age

population affected are commonly between 7 to 78 years , with a male is to

female ratio of 1:1.08. Parotid (56.65%) being the most common organ

involved, followed by submandibular (31.73%) . Neoplastic diseases comprise

75% and non neoplastic disease comprise 25%. Among the salivary gland

neoplasms, benign neoplasms constitute 53.85% ( most common being

pleomorphic adenoma) and malignant neoplasms constitute 21.15%(24).

NON NEOPLASTIC LESIONS

The non neoplastic spectrum of salivary gland lesions include various

infections, inflammations and autoimmune conditions. Some of the conditions

include mucocele, ranula, lymphoepithelial cyst, sclerosing polycystic

adenosis, oncocytic hyperplasia, necrotising sialometaplasia, acute sialadenitis,

chronic sialadenitis, granulomatous sialadenitis and sjogrens syndrome.

17

WHO HISTOLOGICAL CLASSIFICATION OF SALIVARY GLAND

TUMORS

BENIGN EPITHELIAL TUMORS

Myoepithelioma

Pleomorphic adenoma

Warthin tumor

Basal cell adenoma

Sebaceous adenoma

Oncocytoma

Lymphadenoma

Canalicular adenoma

Cystadenoma

Ductal papilloma

MALIGNANT EPITHELIAL

TUMORS

Mucoepidermoid carcinoma

Acinic cell carcinoma

Epithelial – Myoepithelial carcinoma

Polymorphous low grade

Adenocarcinoma

Basal cell adenocarcinoma

Cystadenocarcinoma

Clear cell carcinoma NOS

Sebaceous carcinoma

Sebaceous lymphadenocarcinoma

Mucinous adenocarcinoma

Low grade cribriform

Cystadenocarcinoma

Salivary duct carcinoma

Oncocytic carcinoma

Small cell carcinoma

Adenocarcinoma NOS

Large cell carcinoma

Myoepithelial carcinoma

Lymphoepithelial carcinoma

Carcinoma ex Pleomorphic adenoma

Sialoblastoma

Carcinosarcoma

SOFT TISSUE TUMORS

Hemangioma

HEMATOLYMPHOID TUMORS

Hodgkin lymphoma

Marginal zone B cell lymphoma

Diffuse large B cell lymphoma

18

ETIOLOGY OF SALIVARY NEOPLASMS

The etiological agents causing salivary gland neoplasms are not clear. The

following are few of the factors identified to be associated with salivary gland

neoplasms by various studies

1. Therapeutic irradiation of head and neck region

2. Occupational exposure to rubber manufacturing and wood working

3. Immunosuppression

4. Viruses like EBV and HIV

5. Diet rich in vitamin C and low in cholesterol may be effective in

preventing salivary neoplasms.

BENIGN TUMORS OF THE SALIVARY GLANDS

EPITHELIAL TUMORS

MIXED TUMOR (“PLEOMORPHIC ADENOMA”)

It is the commonest benign tumour of the salivary gland. This tumour

presents as a painless, persistent swelling, and can occur at any age. However,

they most commonly occur in adults during the fourth to sixth decades of

life(25). Rarely, they may be found during childhood, may be bilateral and even

familial mixed tumours have been reported . The most common site of origin is

parotid gland followed by submandibular gland . Its occurrence in the minor

salivary glands is approximately 10%(26). Occasionally, mixed tumours arise in

intraparotid or periparotid lymph nodes or in the heterotopic salivary gland

tissues .The vast majority of mixed tumours arise in the superficial portion of

the parotid gland(27), whereas the rest present in the deep lobe occupying the

parapharyngeal space.

19

On gross examination, the mixed tumour is composed of nodules

connected by a delicate fibrous connective tissue. The tumour is often

multifocal. After wide local excision, the recurrence rate was 4% and 8%

repectively after 1 and 5 years of treatment(28).

Figure 6: Macroscopic picture of pleomorphic adenoma showing well

circumscribed margins

20

Figure7 : Benign mixed tumour showing glandular and chondroid areas,

H&E,4x

WARTHIN TUMOR

Warthin tumour otherwise called as the “papillary cystadenoma

lymphomatosum”, represents about 4 to 10 %(29) of epithelial salivary gland

tumours. It almost exclusively arises from the parotid gland and periparotid

lymph nodes due to the unique admixture of salivary and lymphoid elements in

the parotid region during embryologic development.

Warthin tumour is strongly associated with smoking (30). It commonly

affects men between 60 to 70 years. About 7 to 10 % of patients show

multifocal and bilateral involvement(31).

21

Warthin tumour is well defined and on an average measures 2cm in

greatest dimension. The cut surface is frequently cystic. The cyst contents may

be mucinous or proteinaceous. Some of the lesions are tiny and are incidentally

seen in parotid resections for unrelated reasons. The histologic appearance of

Warthin tumor is very distinctive . It has an epithelial and lymphocytic

component. The epithelial component is composed of oncocytic cells

characterised by their dense granular, eosinoplilic cytoplasm and the lymphoid

component has prominent lymphoid follicles. The oncocytic epithelial elements

have their characteristic cytoplasm as a result of large numbers of mitochondria.

This component consists of two or more layers of cells, which are arranged in a

papillary configuration protruding into a cystic lumina . The cystic spaces

consists of various elements like crystalline structures, cast-off epithelial cells,

corpora amylacea, or inflammatory cells. The luminal epithelium may also show

focal apocrine or squamous metaplasia(32). The lymphoid stroma is composed of

T and B cells with occasional germinal centers. The lymphoid stroma can serve

as a metastatic site for carcinoma or melanoma, or it may give rise to malignant

lymphomas. Rarely squamous or adeno or undifferentiated carcinoma develop

in the epithelium of a Warthin tumor(33).

22

Figure 8: Warthin tumour showing bilayered epithelium and lymphoid

aggregates,H&E,40x

BASAL CELL ADENOMA

Although it occurs in varying age groups, it most commonly affects men

over 60 years(34). Parotid gland is the most common site of origin , giving rise to

around 70% of tumours, with the remainder arising in the submandibular gland

and a variety of other sites like the oral cavity, including the upper lip(35).

Grossly, these tumours are sharply circumscribed and encapsulated masses.

Microscopically, uniform basal cells are arranged in solid, trabecular or

tubular pattern. . A distinct capsule is present. Presence of a distinct capsule

helps in distinguishing basal cell adenoma from adenoid cystic carcinoma and

basal cell adenocarcinoma (35). The tumour cells have a well defined basement

23

membrane that separates it from the non mucoid stroma . Peripheral cell

palisading is a characteristic feature and is best seen in the larger nests of the

solid pattern. The cells within the more central portions of the solid nests often

show squamoid differentiation ranging from only slightly more prominent

eosinophilic cytoplasm to well differentiated forms with keratin pearl formation.

Mitotic count ranges from 0 to 1 per 10 high power field(36).

Immunohistochemically, the tumour cells of basal cell adenoma are

reactive for epithelial membrane antigen (EMA), carcinoembryonic antigen

(CEA), cytokeratin, and, focally, S-100 protein(37) .

Figure 9 : Basal cell adenoma, H&E, 10x

24

CANALICULAR ADENOMA

Canalicular adenoma is closely related to basal cell adenoma. It

commonly affects the adults and most of the time occurs in the seromucinous

glands of the upper lip(38). Grossly, they appear as a single encapsulated nodule,

or a well-defined but unencapsulated nodule, may occasionally show

multinodular growth.

Microscopically, the tumour has tubules or ducts lined by small cuboidal

to columnar cells having scant to moderate amounts of eosinophilic cytoplasm,

resembling basaloid cells. Occasional presence of mucous or oncocytic cells,

papillary structures and psamomma bodies have also been described. The cells

are arranged in cords having intermittent expansions with a canal like lumen .

The stroma is loose and has a myxoid quality with prominent vascularity.

Immunohistochemically, Canalicular adenomas are positive for

cytokeratin and S-100 protein and, focally, for glial fibrillary acidic protein

(GFAP) , SOX10, CAM5.2, CD15(38).

25

Figure 10: Canalicular adenoma showing tubules with canal like lumen ,

H&E,10x

ONCOCYTOMA

Oncocytic cells are cells with abundant, granular, eosinophilic cytoplasm

owing to the presence of numerous mitochondria seen in a wide variety of

salivary neoplasms like mixed tumor, Warthin tumor, acinic cell carcinoma

mucoepidermoid carcinoma and many other benign and malignant lesions. They

are also frequently present in nonneoplastic conditions including incidental foci

of oncocytic metaplasia and oncocytic cysts of the seromucinous glands,

particularly in the false vocal cords(39).

Oncocytomas are benign tumors of salivary glands. They are entirely

composed of oncocytic cells, and they account for 0.1 to 1.4% of all salivary

gland neoplasms(40) . They are tumors of old age, with a mean age of occurrence

of 53 years(41). The most common site of occurrence is parotid, followed by

26

the submandibular salivary gland and then by the minor salivary glands of the

palate, pharynx, lower lip and oral mucosa. History of radiation exposure to the

face or upper body is seen in 20% of all patients(42).

On gross examination, oncocytomas are usually of 3 to 4 cm in size and

have a well-defined capsule, and the cut section has a light brown to

mahogany colour(43) . Microscopically, the oncocytic cells are arranged in solid

sheets , trabecular pattern and rarely microcyst formation is observed.

Occasionally clear cells are seen in oncocytomas. Clear-cell oncocytoma are

tumors that are predominantly composed of clear cell components.

Histochemically the cells of oncocytoma show cytoplasmic positive

staining for phosphotungstic acid hematoxylin (PTAH)(44). By electron

microscopy, the mitochondria are filled with elongated cristae and has an

internal structure that is partially lamellar(45). The nuclei of the oncocytes are

irregular. It contains glycogen granules and inclusions.

Immunohistochemically they show positivity for CK7, CK20, Epithelial

membrane antigen and CD 10 negative(46).

Recurrence in Oncocytomas are very rare(47). The differential diagnosis of

oncocytoma includes the extremely rare oncocytic carcinoma discussed later and

a many other salivary gland lesions that can have at least a minor oncocytic

component. Oncocytomas should have a monomorphous appearance except for

occasional clear cell change.

27

Figure 11: Oncocytes with abundant granular eosinophilic cytoplasm,

H&E, 40x

MYOEPITHELIOMA

These are rare salivary gland tumors accounting to 1 to 1.5% of all

salivary gland neoplasms(48). The lesions present as slow growing,

asymptomatic mass. It arises in a mean age group of 50 years, affecting both

sexes equally. Approximately 50% of myoepitheliomas arise from the parotid

gland, lesions in minor salivary glands constitute 40% and a few from the

submandibular glands(48). These are benign salivary gland tumors composed

entirely of myoepithelial cells. They are encapsulated or sharply demarcated

tumors composed of several distinct cell types, including spindle cells,

plasmacytoid (hyaline) cells and clear cells. Mixtures of cell types in a single

tumor are common.

Cells with myoepithelial differentiation are found in a number of

salivary gland neoplasms, and immunohistochemical markers for this

28

differentiation (e.g., p63, cytokeratin, S100, SMA etc.(48)) can be valuable in

differentiating tumors with myoepithelial cells.

Figure 12: Plasmacytoid myoepithelioma, H&E, 40x

SEBACEOUS LESIONS OF SALIVARY GLANDS

Sebaceous lesions are commonly found in the Parotid (10 to 40 %) and

frequently in submandibular glands (5 to 6 %)(49). They are classified into 5

groups on histological basis

a)SEBACEOUS ADENOMA:

Sebaceous adenoma is a rare disease which constitutes 0.5% of salivary

neoplasms and affects relatively older age group (5th to 6th decade ) having a

male is to female ratio of 4:3(49). Microscopically they are predominantly cystic,

with epithelial cells showing broad areas of sebaceous differentiation with

occasional areas of oncocytic metaplasia(50).

29

b)SEBACEOUS LYMPHADENOMA

It is also a rare benign neoplasm occurring over 50 years of age. Histologically

it shows islands of epithelium showing sebaceous differentiation admixed with

hyperplastic lymphoid tissue(51).

c)SEBACEOUS CARCINOMA

It is a rare and aggressive neoplasm. It is associated with Muir-Torre

syndrome and Familial retinoblastoma. Histologically it shows unencapsulated

and lobular collections of sebaceous and undifferentiated cells(52).

d)SEBACEOUS LYMPHADENOCARCINOMA

The are malignant counterpart of sebaceous lymphadenoma.

Figure13: Sebaceous adenoma of salivary gland, H&E, 40x

DUCTAL PAPILLOMAS

The term ductal papilloma includes three related tumours commonly

arising from the minor salivary glands. It includes sialadenoma papilliferum,

inverted ductal papilloma and intraductal papilloma(53) . These occur in the age

30

group between sixth to eighth decades of life with an average age of 54 years,

and are slightly more common in men(54). Sialadenoma papilliferum commonly

arises from the minor salivary glands of the palate. It has an exophytic surface

and is usually mistaken clinically for a squamous papilloma. Microscopically, it

has a cauliflowerlike surface that merges at its base with underlying often

cystically dilated minor salivary gland ducts . The lining of the complex

papillary structures range from columnar glandular epithelium to keratinizing

squamous cells. A mild to intense mixed inflammatory infiltrate is located in the

fibrovascular cores of the papilla. It has a recurrence rate of 10 to 15 %(54).

Inverted ductal papilloma is a tumour having a endophytic growth that is

contiguous with the surface epithelium but does not extend above it . It consists

of a mixture of squamous and basaloid cells, in a ribbonlike growth pattern, that

projects into a cystic space, representing a dilated salivary gland duct.

Intraductal papilloma is composed of complex branching papillary fronds

within a cystic expansion of a salivary gland duct. It is lined by predominantly

columnar or cuboidal epithelium with scattered mucous cells .

MALIGNANT SALIVARY GLAND NEOPLASMS

MUCOEPIDERMOID CARCINOMA

Mucoepidermoid carcinoma is the third most common minor salivary

gland tumour (15%). They constitute 30 % of all malignant neoplasm of

salivary glands(56). Its incidence peaks between 3rd to 5th decade and is also seen

in childhood. It has a male is to female ratio of 3:2(56).

31

The low-grade tumours are well-circumscribed and are often cystic .

Slow painless growth is a characteristic feature. High-grade tumours are poorly

delineated, solid masses that may be fixed to the surrounding soft tissues. They

are often painful as a result of facial nerve involvement.

On gross examination, the lesions vary from smooth to irregular contours

and measure on an average 3 to 5 cm in size. It consist of both solid and cystic

areas . The cystic spaces may be filled with mucus or hemorrhagic material. On

histologic examination multiple cell types are seen. The most common are

squamous cells, mucous cells, cuboidal intermediate cells, and basaloid cells.

The squamous cells are seen as solid nests, often with individual cell

keratinization and intercellular bridging. This component often predominates in

higher grade tumours. The mucous cells may be diffusely dispersed, or they may

form small clusters partly lining the cystic spaces. Mucous cells are

predominantly seen in low-grade tumours. If the mucinous secretions escape

into adjacent salivary gland tissues, they may elicit a foreign body giant-cell

reaction, that give rise to diagnostic difficulty.

Occasionally prominent clear cells or oncocytic cells may be seen. In low-

grade mucoepidermoid carcinomas, well-formed glandular or microcystic

structures are present; they are lined by a single layer of columnar cells that

secrete mucin. In some areas, these cystic spaces are bordered by papillary

infoldings that are formed by intermediate, basaloid, or squamous cells. In these

low-grade malignant lesions, microcysts that coalesce into macrocysts may be

quite prominent.

32

Intermediate-grade malignant lesions are usually characterized by solid

areas of squamous, intermediate, basaloid cells or clear cells or by papillary

cystic infoldings.

High grade tumours are characterised by variations in the shape and size

of the carcinoma cells, prominent nucleoli, and abundant mitotic figures .

Obvious invasion, including perineural extension, focal necrosis, increased

mitotic figures, and greater pleomorphism are also more evident .

Immunohistochemically mucoepidermoid carcinomas express a keratin

profile of CK7+, CK14+, CK20- and a mucin profile showing positivity for

MUC1, MUC2, MUC4, MUC5AC,MUC5B and negative for MUC3.

Figure 14: Mucoepidermoid carcinoma, H&E,10x

33

ADENOID CYSTIC CARCINOMA

This tumour comprises about 10% of all salivary neoplasms. It occurs

between fifth and sixth decades with a female preponderance(56). It is the most

common malignant tumour of the submandibular glands and minor salivary

glands .

This tumour grossly appears as a solid and circumscribed mass, but

microscopically, it extends beyond the grossly visible and palpable limits of the

lesion. Three histologic patterns are typically observed in these tumours which

include cribriform, tubular, and solid. Multiple patterns are often present in a

single tumour. The cribriform, or classic, pattern consists of basaloid epithelial

cells forming sharply demarcated nests containing multiple extracellular spaces.

The spaces contain PAS-positive connective tissue mucin or eosinophilic

hyaline-like material.

The infiltrative capacity of this tumour is the hallmark of this salivary

gland carcinoma. It spreads along nerve sheaths and is associated with severe

pain. The lesion commonly presents as Facial nerve paralysis ( may be the

presenting symptom) , and is associated with multiple late local recurrences

after operative intervention .

This malignant tumour occasionally spreads to regional nodes, but

hematogenous tumour spread is often seen in the lungs. Though the lung

metastasis is clearly identifiable on routine chest radiographs, it may remain

stable for many years. Immunohistochemically, adenoid cystic carcinomas,

34

show strong positivity for C-KIT, CK7, CAM5.2, calponin, SMA, p63, S100 and

SOX10(57).

Figure 15:Adenoid cystic carcinoma , H&E, 40x

ACINIC CELL CARCINOMA

It is an uncommon tumour of salivary gland, occurring more commonly

in women, having peak incidence in the fifth and sixth decades of life(58). It

represent approximately 7 to 15% of all malignant salivary gland tumours(58),

with about 90% of these tumours arising in the parotid gland(81 to 98%)(59).

Previous history of radiation exposure and family history are predisposing

factors. It often presents as a slow growing mass of parotid.

On gross examination, acinic cell carcinoma are often well delineated,

solid, and yellow, gray, or brown in color with occasional grossly visible cysts .

35

Microscopically, the acinar tumour cells closely resemble the serous

cellular elements of the normal parotid gland with finely granular, basophilic

cytoplasm . In addition to the classic serous cells, various other cell types seen

are clear cells, oncocytic cells, basaloid cells, and nonspecific-appearing ductal

cells, arranged in equally varied growth patterns, including solid, acinar,

microcystic, papillary cystic, and follicular. Acinic cell carcinoma is a well-

differentiated tumour. It typically lacks the overt cytologic features of

malignancy. Dedifferentiated acinic cell carcinoma is a highly aggressive tumour

that requires adjuvant treatment .

Acinic cell carcinoma has a tendency for recurrence (35%) and distant

metastasis especially to lungs and regional nodes(59).

Figure 16:Acinic cell carcinoma with intensely basophilic cytoplasm,

H&E, 10x

36

POLYMORPHOUS LOW-GRADE ADENOCARCINOMA

(PLGA, lobular carcinoma, terminal duct carcinoma, trabecular

carcinoma). This tumour exclusively involves the minor salivary glands,

particularly those of the palate(32%), buccal mucosa(10%) and upper lip. This

tumour is twice more common in women when compared with men, and it

affects age group between 23 to 94 years(60) .

These tumours present as slowly enlarging, painless palatal masses or

swellings (49%). Before the recognition of PLGA, this tumour was

misdiagnosed as adenoid cystic carcinoma. It may also involve the buccal

mucosa, lip, retromolar triangle, cheek, and tongue. Involvement of major

salivary glands is extremely rare and always occurs in the parotid gland in the

setting of a carcinoma arising in a pre-existent mixed tumour.

These tumours range in size from 1 to 5 cm and usually have intact

overlying mucosa. These tumors are grossly well delineated lesions but lack

capsule formation. The cells are monomorphous, are of cuboidal or low

columnar type with oval or elongated uniform basophilic nuclei. The cytoplasm

is modest in amount and usually eosinophilic. Intralesional necrosis and mitotic

figures are rare.

Immunohistochemical studies show consistent reactivity to EMA and

more variable staining for CEA in PLGA. It is a low grade tumour with distant

metastasis of <1%(61).

37

SALIVARY DUCT CARCINOMA

It is an aggressive neoplasm constituting 1 to 3% of all malignant

salivary gland tumours, predominantly of middle-aged and elderly males(55 to

61 years),occurring primarily in the parotid gland (80%)(62). Clinically

patients present with a mass, and many have facial nerve involvement.

Histologically this tumour predominantly resembles ductal carcinoma breast.

Variants of this tumour include Mucinous , micropapillary and sarcomatoid .

The micropapillary variant has been noted to have an extremely poor prognosis,

and the sarcomatoid variant may be difficult to differentiate from a

carcinosarcoma. Salivary duct carcinoma is positive for human epidermal

growth factor 2 (Her2), CK 8/CK18,p63,calponin and negative for estrogen and

progesterone receptor. It behaves aggressively and has a tendency for early

cervical lymphadenopathy and distant metastasis to lungs and bones(63). It is

treated with radical excision and lymphadenectomy with radiation therapy or

chemotherapy.

MALIGNANCY ASSOCIATED WITH MIXED TUMOUR

It includes three clinicopathological entities - benign metastasizing

mixed tumour, salivary carcinosarcoma or true malignant mixed tumour and

carcinoma arising in a mixed tumour. It constitutes about 3.6% of all salivary

gland neoplasms and 6.2% of all benign mixed tumours(64).

Benign Metastasizing Mixed Tumour is the term applied to entirely

benign looking mixed tumours that show metastasis. Most common site of origin

38

is parotid. The sites of metastases are most commonly bone and lungs. Almost

all patients with benign metastasizing mixed tumours have an entirely benign

clinical course. Very rare aggressive behaviour has also been noted.

Carcinoma arising in Mixed tumour are mixtures of benign mixed tumour

and a distinct second component of carcinoma . The clinical presentation follows

one of the three pathways. Most commonly, an asymptomatic salivary gland

swelling, presumably the benign component, is present for many years and then

suddenly enlarges and becomes painful, signaling the development of the

malignant component with neural invasion. Alternatively, following one or more

local recurrences of a benign mixed tumour, the subsequent recurrence consists

of a carcinoma. Residual benign mixed tumour may or may not be identifiable.

Finally, salivary gland symptomatology may be noted for less than a year, and

the resection documents a benign mixed tumour with associated carcinoma, with

both components apparently originating almost simultaneously.

39

Figure 17 : Carcinoma ex pleomorphic adenoma showing (A) chondroid

(B) malignant areas,H&E, A-4x, B-40x

EPITHELIAL-MYOEPITHELIAL CARCINOMA

It represent <1% of all salivary gland neoplasms. It commonly occurs in

the 7th decade of life with a female predominance(65). Parotid gland is the most

common site of occurrence , with the rest occurring in the submandibular and

minor salivary glands.

On histologic examination, a well-defined fibrous capsule surrounds a

multinodular growth pattern , The capsule may be focally absent or may be

breached by nests of tumour cells. In the classic form of this tumour, two tumour

40

cell types are present in a characteristic biphasic appearance. Small cuboidal

cells with amphophilic cytoplasm and eccentric small nuclei form duct like

structures with central lumen formation. These ductal epithelial cells contain

PAS-positive material that is not removable by diastase. Surrounding these cells

is a layer of larger oval or polyhedral myoepithelial cells with clear cytoplasm on

H&E-stained sections. PAS-positive basement membrane may be seen

ensheathing tumour cell nests in an organoid growth pattern.

Immunohistochemically the ductal epithelial cells react for cytokeratin,

EMA, and occasionally S-100 protein. The clear myoepithelial cells will

demonstrate variable cytokeratin reactivity with strong reactivity for S-100

protein, smooth muscle actin, p63, CD10, calponin, and other myoepithelial

markers.

Regional lymph node metastases and distant blood-borne metastatic

spread to lungs and kidneys have been reported with a

recurrence rate of 23 to 50%(65).

BASAL CELL ADENOCARCINOMA

Basal cell adenocarcinoma is the malignant counterpart of basal cell

adenoma. It differs from basal cell adenoma by the presence of capsular and

stromal invasion. This tumour accounts for about 2% of salivary malignancies

and occurs predominantly in the parotid gland of elderly individuals (median

age, 60 years) . Local recurrences, regional lymph node metastases, and

pulmonary metastases have all been reported. Basal cell adenocarcinoma appears

41

to be a low-grade, favourable malignancy that must be distinguished from more

aggressive salivary neoplasms such as adenoid cystic carcinoma on the one hand

and basal cell adenoma on the other hand. Immunohistochemically it shows

positive staining for S100 and Cytokeratin 7 (66) .

ONCOCYTIC CARCINOMA

Oncocytic carcinoma is an extremely rare, aggressive, infiltrative

neoplasm accounting for 0.5% of epithelial salivary gland malignancies(67). They

resemble benign Oncocytomas microscopically and require the following criteria

for diagnosis – a) local nodal metastasis, b)distant metastasis,

c)perineural,vascular or lymphatic invasion d)frequent mitosis and cellular

pleomorphism. Survival period on an average is 3.8 years with metastasing

tumours(67).

Figure 18: Oncocytic carcinoma showing oncocytic cells and mitotic figures,

H&E,40x

42

SEBACEOUS CARCINOMA

Sebaceous carcinoma is a rare neoplasm. They are the malignant

counterpart of salivary adenoma. It is distinguished by its cellular atypia and

stromal invasion. The treatment often requires total parotidectomy and adjuvant

radiation therapy.

MYOEPITHELIAL CARCINOMA

Myoepithelial carcinoma, the malignant counterpart of myoepithelioma, It

is a rare neoplasm that constitutes about 1% of all salivary neoplasms(68) and it

occurs primarily in the parotid. The mean age of occurrence is 55 years with a

higher incidence in women.

It is characterized grossly by multinodularity, necrosis and lack of

encapsulation. The histologic features essential to diagnose myoepithelial

carcinoma are the presence of exclusive myoepithelial differensiation , cytologic

atypia and infiltration into adjacent structures. Myoepithelial carcinoma is

composed of varying proportions of spindled, epithelioid, plasmacytoid, and

clear cells set against a mucoid or myxoid background . The majority of the

tumour express myoepithelial differentiation by either immunohistochemistry or

electron microscopy.

Immunohistochemical confirmation is required. They are positive for

vimentin, cytokeratins, S-100, Calponin, EMA, p63, CD10 and negative for

CEA(69). They exhibit variable behaviour with 40% cases showing local

recurrence and 30% showing postoperative metastasis(69).

43

LYMPHOEPITHELIOMA-LIKE UNDIFFERENTIATED CARCINOMA

It is an an undifferentiated carcinoma that is morphologically

indistinguishable from a nasopharyngeal lymphoepithelioma . It commonly

arises from the Parotid . This tumour is particularly common in native Alaskan

Eskimo and Orientals(70). The patients may range in age from 2nd to 6th decade.

As with its nasopharyngeal counterpart, this tumour is strongly associated with

EBV(70).

On histologic examination, neoplastic epithelial cells either singly or in

nests or islands are seen admixed with diffuse lymphoplasmacytic infiltrate. At

higher magnification, the neoplastic cells are clearly malignant with considerably

enlarged but morphologically and chromatically uniform, ovoid to elongated

vesicular nuclei and variably prominent nucleoli . When present in nests, the

cells have indistinct borders such that the vesicular nuclei appear to float in a

cytoplasmic syncytium. Variable numbers of lymphocytes, plasma cells, and

occasionally other inflammatory cells permeate the cell nests. Mitotic figures,

including atypical forms, are numerous. Perineural invasion can be found in

approximately half of cases .

IMMUNOHISTOCHEMISTRY IN SALIVARY GLAND

With the increasing incidence and with improved treatment modalities for

salivary gland tumours it has now become essential to accurately diagnose the

various tumours of the salivary glands. Though H&E staining is the gold

44

standard method to diagnose these tumours, IHC greatly enhances the diagnostic

accuracy as well as guides us through these diagnostic challenges.

IMMUNOHISTOCHEMISTRY

Immunohistochemistry is one of the ancillary techniques in pathology. It

detects antigens in tissue sections by immunological and chemical reactions.

These antigens may be proteins, amino acids , infectious agents or even a

particular cell populations. It thereby aids in diagnosis and prognostication of

various diseases.

It comprises of two phases -1)slide preparation and stages of reaction

2)interpretation and quantification

For a successful and reliable Immunohistochemistry both the above said

steps should be done by following standard protocols.

SLIDE PREPARATION AND STAGES OF REACTION

Optimal tissue fixation ( using 10% Neutral buffered formalin) and

processing are essential and greatly influence the quality of

immunohistochemistry. Tissue sections of 3 to 4 microns are obtained(71) .

Thicker sections may be washed out during antigen retrival and more over

hinder efficient staining. Tissue sections are obtained on coated slides, which are

adhesive slides and are positively charged. Theses slides firmly attach the tissue

proteins that are negatively charged. It is essential for the tissue sections to be

45

firmly adheared to the slides to prevent partial detachment of tissue sections

during staining and there by leading to trapping of staining reagents.

ANTIGEN RETRIVAL:

It is the technique of exposing of antigen epitopes in tissues thereby

favouring the antigen antibody reactions in the consequent steps of the

reaction. It is done by immersing tissue sections in antigen retrival solutions like

Tris- Hcl or sodium acetate buffer at pH 8.0 to 9.0 and exposing them to heat or

enzymes. By exposing the tissues to certain enzymes (Proteinase K, Trypsin,

Pepsin, etc) or by heating the tissues to high temperature ( using microwave,

pressure cooker, waterbath, etc ) the tissue proteins are hydrolysed which breaks

the cross links between various amino acids and peptides thus exposing the

antigen epitopes, for the antibodies to bind.

ANTIGEN – ANTIBODY BINDING

The antibodies may be monoclonal or polyclonal and are commercially

prepared by either immunising an animal with the antigen of interest or by

culturing plasma cells exposed to the antigen of interest in an appropriate

medium.

The antibodies thus obtained, when applied to tissue sections combines

with the epitope of antigens by means of amino acid side chains which are

complimentary to each other.

46

BLOCKING BACKGROUND STAINING:

Background staining is the unwanted staining of cells in the section. The

common causes for background staining in Immunohistochemistry are either non

specific antibody binding to endogenous Fc receptors or due to hydrophobic

and ionic interactions and endogenous enzyme activity. Background staining

may either be specific or non specific. Specific background staining occurs due

to apparent affinity of certain tissue components to the applied antibodies. Non

specific background staining occurs due to the presence of charged sites in the

tissue section , especially the high affinity of collagen and reticulin for

immunoglobulins.

Background staining can be minimised by adding a innocuous protein

solution to the section before applying the primary antibody. The applied protein

should saturate and neutralize the charged sites, thus enabling the primary

antibody to bind to the antigenic site only. Blocking of endogenous enzymatic

activity should be done before addition of enzyme labelled secondary reagent to

prevent the inactivation of enzyme label thereby resulting in a false negative

result.

DETECTION SYSTEMS

One method to identify the antibody bound antigen is using chromogen

3,3’- diaminobenzidine (DAB). It is a horseradish peroxidise (HRP) substrate

that is used for secondary HRP – conjugated antibody detection. In the presence

of hydrogen peroxide (H2O2) and in the presence of enzyme horseradish

47

peroxidise, DAB is converted into an insoluble brown reaction product and

water(72). The coloured product acts as a flag to identify the presence of the

antigen of interest.

DAB + H2O2 ------ > HRP ----------> DAB ppt + H2O

WASHES

To prevent the formation and deposition of antigen antibody complexes

that gives unwanted background staining, it is necessary to remove unbound

antibody in each step. This is done by washing the sections in Tris –buffered

saline (TBS).

APPLICATIONS AND IMPORTANCE OF

IMMUNOHISTOCHEMISTRY

1) Histogenic diagnosis of morphologically non differentiated neoplasia

2) Subtyping of tumours

3) Identification of primary site or tissue of origin of tumours

4) To Discriminate benign from malignant proliferations of certain cell

populations

5) To identify prognostic factors and therapeutic indications of some

diseases.

LIMITATIONS OF IHC

Various factors influence the reliability of immunohistochemistry

performed starting from specimen fixation, tissue processing, antigen retrival,

48

detection system, the panel of antibody selected, choice of antibody types and the

rigor of execution.

Given below is a table of common IHC markers used in tumors of salivary

Glands

Markers[antibodies]

Positivity innormal salivaryglandparenchymalcells

Uses and significance for salivary glandtumors

Pancytokeratin (CK)luminal andabluminal cells

Epithelial marker; used in differentialdiagnosis betweenmyoepithelioma/myoepithelial carcinoma

Carcinoembryonicantigen (CEA)

Luminal cells Ductal (luminal) cell marker

Epithelial membraneantigen(EMA)

Luminal cellsDuctal cell marker, shows apical stainingpattern, exhibits bubbly positive insebaceous cells

CalponinMyoepithelialcells

Myoepithelial marker (high specificity,very useful)

α-Smooth muscleactin (SMA)

Myoepithelialcells

Myoepithelial marker (high specificity,very useful)

49

Markers[antibodies]

Positivity innormal salivaryglandparenchymalcells

Uses and significance for salivary glandtumors

P63Myoepithelialand basal cells

Myoepithelial marker

Also shows positivity for squamous andbasal epithelial cells

Muscle-specific actin(MSA)

Myoepithelialcells

Myoepithelial marker

Highly specific

Glial fibrillary acidicprotein (GFAP)

Myoepithelialcells

Myoepithelial marker (low sensitivity);highly positive in pleomorphic adenomaand myoepithelioma

CK14Myoepithelialcells and basalcells

Myoepithelial marker

Also positive for squamous and basalcells

VimentinMyoepithelialcells

Myoepithelial marker (good forscreening, low specificity)

S-100 protein variableMyoepithelial marker (good forscreening, low specificity)

50

Markers[antibodies]

Positivity innormal salivaryglandparenchymalcells

Uses and significance for salivary glandtumors

Ki-67 [MIB-1] Few cellsCell proliferation marker, to differentiatebetween benign and malignant tumors,also a prognostic factor

p53 NegativeUsed in the differential diagnosisbetween benign and malignant tumorsalso a prognostic factor

HER2/neuNegative toweakly positivein ductal cells

Overexpressed in salivary ductcarcinoma, used in diagnosis of non-invasive carcinoma ex pleomorphicadenoma, expected candidate formolecular targeted therapy

Androgen receptor Negative

Often positive in salivary ductcarcinoma, used in diagnosis of non-invasive carcinoma ex pleomorphicadenoma, expected candidate formolecular targeted therapy

α-Amylase Acinar cellsShows positivity in acinic cellcarcinoma(low sensitivity)

Mitochondria Striated duct cells Strongly expressed in oncocytic cells

51

Markers[antibodies]

Positivity innormal salivaryglandparenchymalcells

Uses and significance for salivary glandtumors

Gross cystic diseasefluid protein-15

Luminal cellsFrequently positive in salivary ductcarcinoma (low specificity)

Melan A NegativeUsed in diagnosis of metastatic renalcell carcinoma

Renal cellcarcinoma(CD10)

NegativeUsed in diagnosis for metastaticmalignant melanoma

Lymphoid cellmarkers

NegativeUsed in diagnosis of malignantlymphoma

EBER in situhybridization

Negative Expressed in lymphoepithelial carcinoma

52

Ki67 AND p63 IN SALIVARY GLAND NEOPLASMS

Ki 67 is large (395KD) ,nuclear, non histone protein(73). It is associated

with cell proliferation. It is encoded by the MKI67 gene(74) . It is associated with

transcription of ribosomal RNA. Unlike other cell cycle associated proteins, it is

consistently absent in quiescent cells and during DNA repair process. It is

expressed in all active phase of cell cycle except in G0 phase ( resting cells) and

so reflects the proliferation status of the cell. It is a typical nuclear stain taken up

by the proliferating cells and as the proliferation status is closely related to

tumour aggressiveness, Ki67 labelling index is an established prognostic

marker for various tumours and is also useful in determining the recurrence rate.

p63 is a member of p53 family genes. Similar to p53, it codes for

proteins having an amino (N) terminal transcription activating region, middle

DNA binding region and terminal carboxy region(75). Previous studies have

shown that p63 is expressed in various normal tissues like the bronchial

epithelium, squamous epithelium, myoepithelial layers of breast and

urothelium. Benign myoepithelial cells of salivary gland and normal prostate

express p63. Its expression has been established in various neoplasms like

squamous cell carcinoma, urothelial carcinoma, papillary thyroid carcinoma,

thymomas and endometrial carcinomas. All salivary gland neoplasms that had

myoepithelial cell differentiation also expressed p63.

It also regulates growth of the salivary glands and it helps in regulation of

differentiation and proliferation in epithelial progenitor cells.

53

MATERIALS AND METHODS

The present study is a retrospective study to assess the expression of

Ki67 and p63 in various benign and malignant salivary gland tumours and to

assess the diagnostic and prognostic significance of these markers in salivary

gland tumours. Approval of ethical committee was obtained to conduct this

research study.

STUDY LOCATION

The study was conducted at the Department of Pathology, Tirunelveli

medical college.

STUDY PERIOD

This study was conducted from June 2015 to June 2016.

INCLUSION CRITERIA

1. All salivary gland neoplasms operated in Tirunelveli medical college

hospital

2. Includes both benign and malignant neoplasms

EXCLUSION CRITERIA

Inflammatory and other non neoplastic diseases of salivary glands

SAMPLE SIZE

A total of 46 cases of salivary gland neoplasms were studied

54

METHODOLOGY

The details of the patient and the histopathological diagnosis were

collected from the general surgical pathology report register, clinical case sheets

and from medical records department of Tirunelveli medical college hospital. A

total of 46 cases of reported salivary gland neoplasms were collected , who

were operated between 2012 and 2016.

The corresponding Pathology numbers of these 46 patients were identified

from Surgical Pathology register and the blocks were collected from the

department of Pathology of Tirunelveli medical college. The collected data were

recorded in a master chart (Annexure -3).

The expression of Ki67 and p63 of the collected cases were assessed by

Immunohistochemistry.

SLIDE PREPARATION FOR IMMUNOHISTOCHEMISTRY

Special positively charged or coated slides are used for IHC. The selected

block was cut using a microtome into 5 micron thick sections and these

sections were transferred on to the coated slides. After mounting, these slides

were dehydrated by keeping in an incubator at 60 degree celcius for 8 hours.

55

BUFFER PREPARATION

TRIS EDTA BUFFER

Tris – 6.05gm

EDTA – 0.744g

1N Hcl – 4ml

Distilled water – 1 litre

Required pH - 9

PRECAUTIONS

1. The glassware used in IHC must be dry and clean.

2. The buffer used should be freshly prepared and preferred ph must be

achieved by adjusting accordingly.

3. During the process of IHC, the slide should never be allowed to dry, and

so a humidity chamber is used during staining and incubation.

4. DAB chromogen should be carefully handled as it is a potentially

carcinogenic agent.

5. All reagents - Primary antibody, DAB chromogen, Peroxidase block ,

should be stored in an ideal 4-6 degree celcius.

6. Every batch of slide should be accompanied by a positive and negative

control which ensures the quality of the procedure.

56

PROCEDURE

1.Deparaffinization – slides are deparaffinised by placing in xylene

2.The slides are hydrated in graded solutions of alcohol and brought to water

3.Retrival of primary antibody – done in TRIS – EDTA buffer , heat is used for

antigen retrival (using a induction hot plate or a microwave)

3.Application of Primary antibody and incubation

4.Peroxidase block and DAB chromogen is applied to enable viewing by light

microscopy

5.Then the slides are counterstained by Mayers Hematoxylin

6. Consequently the slides are dehydrated in increasing concentration of alcohol

Ki67 SCORING :

Cells with brown staining of nucleus and nuclear membrane were taken as

positive cells, the cells with inconspicuous nuclei were not accounted.

Interpretation of Ki 67 staining was done based on previous studies(76).

In the present study, three areas of the studied tissue with highest density

of tumour cells expressing Ki67 was selected using low power . 1000 malignant

cells from these areas were counted manually and the following formula was

applied to calculate the mitotic index

57

MI = n x 100

1000

MI – mitotic index

n- number of tumour cells positive for a total of 1000 counted cells

0- <1% - score 0

2– 5 % - score 1

>5% - score 2

p63 GRADING :

Interpretation of p63 was done based on previous studies(77).

Only nuclear reactivity was considered positive.

p63 grading is as follows (Nermine M Abd & Sarah A Hakim)

<10% - negative

10 – 25% - weak positive

26 – 75% - moderate positive

76 – 100% - strong positive

The results were analyzed, tabulated and compared with the existing

literature.

58

OBSERVATION AND RESULTS

This study constituted a total of 46 cases of salivary gland neoplasms

which includes both benign and malignant lesions. All the cases were inpatients,

admitted and operated in Tirunelveli Medical College hospital during the 5 year

period between 2012 to 2016.

The age of occurrence of these tumours ranges from 14 years to maximum

of 71 years. The mean age of occurrence was 49.5 years. Of the total 46 cases ,

22 were males (47.8 %) and 24 were females (52.17 %) .The distribution of

lesion in various salivary glands was 32 (69.56 %) in parotid, 10 (21.7 %) in

submandibular, 4(8.69 %) in other minor salivary glands.

Out of the 46 cases , 35 (76.08 %) were benign and 11 (23.91 %) were

malignant. The age group affected by benign tumours was between 14 and 64

years and mean age of occurrence was 39 years.

The most common benign tumour in the present study was pleomorphic

adenoma - 29 cases (63.04 %). It had a mean age of occurrence of 35.44 years

and a male: female ratio of 1:1.9 . The most common site of occurrence was

parotid (55.17 %) , followed by submandibular ( 37.93 %) and other minor

salivary glands (6.8%). Pleomorphic adenoma shows significant statistical

difference in relation to sex, age and site of occurrence.

In this study, malignant tumours generally affected age group from 26

to 71 years, with a mean age of occurrence of 48.09.

59

The commonest malignant neoplasm seen in the study was

mucoepidermoid carcinoma – 7cases (15.21 %). The mean age of occurrence

was 47 years. It has male: female ratio of 2.5:1

The second most common salivary gland neoplasm was acinic cell

carcinoma -2 cases (4.34 %) .

Cases of Warthins tumour (2), Basal cell adenoma (2), Acinic cell

carcinoma (2), oncocytoma (1), malignant oncocytoma (1), salivary duct

carcinoma(1) were all included in the study.

Ki67 expression was studied for all the 46 cases which showed positivity

for 10 cases (21.73 %), out of the 10 cases, 5 cases (14.28 % of benign

tumours) were benign and 5 cases (45.45 % of malignant tumours) were

malignant. Ki67 expression showed significant statistical difference in relation

to benign and malignant tumours.

P63 expression was studied for 30 cases which included 11malignant

cases and 19 benign cases. Out of the 11 malignant cases 3 were negative. Out

of the 19 benign cases studied all showed positivity for p63 ranging from weak

positive to strong positive.

60

TABLE 1: DISTRIBUTION OF CASES

NO OF CASES PERCENTAGE

BENIGN 35 76.08

MALIGNANT 11 23.91

CHART 1: DISTRIBUTION OF CASES

DISTRIBUTION OF CASES

BENIGN

MALIGNANT

61

CHART 2 :AGE WISE DISTRIBUTION OF BENIGN CASES

CHART 3: AGE WISE DISTRIBUTION OF MALIGNANT CASES

0

2

4

6

8

10

12

14

16

18

0 t0 20 yrs 20 to 40 yrs 40 to 60 yrs

0

1

2

3

4

5

6

7

0 to 20 yrs 20 t0 40 yrs 40 to 60 yrs >60yrs

AGE

62

TABLE 2 : DISTRIBUTION OF BENIGN CASES

CASES NUMBER

PLEOMORPHIC ADENOMA 29

MONOMORPHIC ADENOMA 1

ONCOCYTOMA 1

WARTHINS TUMOR 2

BASAL CELL ADENOMA 2

TABLE 3: DISTRIBUTION OF MALIGNANT CASES

CASES NUMBER

MUCOEPIDERMOID CARCINOMA 7

MALIGNANT ONCOCYTOMA 1

ACINIC CELL CARCNOMA 2

SALIVARY DUCT CARCINOMA 1

63

TABLE 4: Ki67 EXPRESSION OF VARIOUS CASES

CASES NO OF Ki67 POSITIVE

PLEOMORPHIC ADENOMA 4

MONOMORPHIC

ADENOMA

NIL

WARTHINS NIL

BASAL CELL ADENOMA NIL

ONCOCYTOMA 1

MUCOEPIDERMOID

CARCINOMA

3

ACINIC CELL CARCINOMA NIL

MALIGNANT

ONCOCYTOMA

1

SALIVARY DUCT

CARCINOMA

1

64

TABLE 5: COMPARISON OF Ki67 EXPRESSION

CASES TOTAL CASES Ki67 POSITIVE %POSITIVE

BENIGN 35 5 14.28%

MALIGNANT 11 5 45.45%

P = 0.028805

From the above table we can see that, expression of Ki67 in malignant

salivary neoplasms is significantly higher than in benign tumors.

CHART 4 : Ki 67 EXPRESSION OF SALIVARY GLAND NEOPLASMS

Ki67 EXPRESSION

BENIGN

MALIGNANT

NEGATIVE

65

TABLE 6: p63 EXPRESSION IN VARIOUS CASES

CASE TOTAL NO OF CASES TOTAL NO OF

POSITIVE CASES

PLEOMORPHIC

ADENOMA

15 15

WARTHINS TUMOR 2 2

BASAL CELL

ADENOMA

2 2

MUCOEPIDERMOID

CA

7 3

ACINIC CELL

CARCINOMA

2 0

MALIGNANT

ONCOCYTOMA

1 0

SALIVARY DUCT

CARCINOMA

1 0

TABLE 7: COMPARISON OF p63 EXPRESSION

CASES TOTAL CASES P63 POSITIVE %POSITIVE

BENIGN 19 19 100%

MALIGNANT 11 3 27.27%

From the above table we can see that p63 expression is significantly higher in

benign tumours when compared to malignant salivary neoplasms.

66

TABLE 8:Ki67 GRADING OF BENIGN SALIVARY TUMORS

CASE TOTALNO

0 1+ 2+

PLEOMORPHC

ADENOMA

29 25 3 1

MONOMORPHC

ADENOMA

1 1 nil nil

ONCOCYTOMA 1 nil 1 nil

WARTHINS 2 2 nil nil

BASAL CELL

ADENOMA

2 2 nil nil

TABLE 9: Ki67 GRADING OF MALIGNANT SALIVARY TUMORS

CASE TOTAL

NO

0 1+ 2+

MUCOEPIDERMOID

CARCINOMA

7 4 2 1

ACINIC CELL

CARCINOMA

2 2 nil nil

MALIGNANT

ONCOCYTOMA

1 nil nil 1

SALIVARY DUCT

CARCINOMA

1 nil nil 1

67

TABLE 10: p63 GRADING OF BENIGN TUMORS

CASETOTAL

NONEGATIVE WEAK MODERATE STRONG

PLEOMORPHIC

ADENOMA

15 nil 2 9 4

WARTHINS 2 nil 1 1 nil

BASAL CELL

ADENOMA

2 nil 2 nil nil

TABLE 11: p63 GRADING OF MALIGNANT SALIVARY TUMORS

CASE TOTAL

NO

NEGATIVE WEAK MODERATE STRONG

MUCOEPIDERMO

ID CARCINOMA

7 4 3 nil nil

ACINIC CELL

CARCINOMA

2 2 nil nil nil

MALIGNANT

ONCOCYTOMA

1 1 nil nil nil

SALIVARY DUCT

CARCINOMA

1 1 nil nil nil

68

CHART 5: Ki67 AND p63 EXPRESSION OF BENIGN TUMORS

CHART 6:Ki67 AND p63 EXPRESSION OF MALIGNANT NEOPLASMS

0

2

4

6

8

10

12

14

16

18

PLEOMORPHICADENOMA

MONOMORPHICADENOMA

WARTHINS BASAL CELLADENOMA

ONCOCYTOMA

Ki67

P63

0

0.5

1

1.5

2

2.5

3

3.5

MUCOEPIDERMOIDCARCINOMA

ACINIC CELLCARCINOMA

MALIGNANTONCOCYTOMA

SALIVARY DUCTCARCINOMA

Ki67

p63

69

Ki67 EXPRESSION IN PLEOMORPHIC ADENOMA

STUDY TOTAL CASES Ki67 POSITIVE

Present Study 29 4

Tadbir et al 20 10

Bhupesh Bhayyaji et al 20 8

Alexandra Corna et al 8 4

Alves Fa et al 15 0

P value - 0.002124

From the above table we infer that the expression of Ki67 in

Pleomorphic adenoma is statistically significant, which is further discussed later.

70

DISCUSSION

Salivary gland tumours are rare entities with an incidence of 2.5 to 3 per

100,000 per year(78). 80% of salivary gland tumours are benign. Incidence of

malignant salivary gland neoplasms range from 0.5 to 2 per 100,000 population

per year(79). Of the benign salivary neoplasms, the commonest is pleomorphic

adenoma which accounts for 70% of benign salivary neoplasms(80).

Mucoepidermoid carcinoma is the commonest of the malignant salivary

neoplasms(81).

In our study, 35 out of the 46 salivary tumours were benign, constituting

76.08% and the remaining 23.91% were malignant tumours. Pleomorphic

adenoma was the commonest of all salivary neoplasms and it constituted

63.04% of all tumours, the second commonest was mucoepidermoid carcinoma

constituting 15.21% of all salivary tumours and our observations were in

concordance with the literature.

Salivary gland neoplasms were more frequent in women with a

male:female ratio of 1:1.6. Benign tumors of the salivary gland were commonly

observed during the third to fourth decade of life and malignant tumors occurred

at the sixth decade(82).

In the present study, the age group affected by salivary neoplasms were

between 14 to 71 years, with a male : female ratio of 1:1.09. Benign tumours had

a mean age of occurrence of 33.6 years and malignant tumours were seen to

71

have a mean age of 50.3 years and these data were seen to correlate with the

above studies.

Various studies referred with regard to site of occurrence of salivary

gland neoplasms reveal that 70 to 80% of cases occur in the parotid gland.

Articles by many authors reveal submandibular salivary gland to be the second

most common site of occurrence while others consider minor salivary glands to

be the 2nd most commonest site(83,84). In our study, Parotid was the most common

gland affected (69.5%) followed by submandibular (21.7%) and other minor

salivary glands(8.69%).

Salivary gland tumours with their diverse morphology , are a source of

diagnostic challenge to the pathologist. IHC plays a significant role both in

aiding the diagnosis of challenging cases and as a prognostic marker of salivary

gland neoplasms.

Ki67 is a proliferative marker used to assess the proliferative potential of

a malignancy. It is a monoclonal antibody that particularly reacts with the

proliferating cells, except those cells in G0 phase of cell cycle. The strong

association between the frequency of Ki67 positivity and higher degree of

malignancy has been well established by various studies for various

malignancies.

In our study, out of 35 benign neoplasms, 4 were positive for Ki67. All

four were pleomorphic adenomas. According to one study, conducted by Mioara

72

Trandafirescu et al (2012)(85), Ki67 positivity in pleomorphic adenoma is taken

as predictor of malignancy risk.

In another study conducted by Anna Kazanceva et al in 2011(86), Ki67

expression was higher in recurrent pleomorphic adenomas than in primary

tumours. The above mentioned 4 cases of pleomorphic adenoma which showed

Ki67 positivity, have been kept under follow up to detect any recurrence or

malignant transformation.

According to the study by Mioara Trandafirescu et al loc.cit in 2012,

done for various immunohistochemical profile of pleomorphic adenoma on 30

cases, Ki67 showed a positive reaction in myxoid areas and in peripherally

situated myoepithelial cell layers. Moreover according to the study, intensity of

Ki67 reaction was higher in Carcinoma ex Pleomorphic adenoma. The study

also states that Ki67 marker is useful in assessing the intensity of proliferation in

cases of Pleomorphic adenoma and thereby gives indications of risk of

malignancy.

None of the other benign tumours in our study showed positivity for Ki67

staining . In Warthins tumour the germinal centre lymphoid cells showed

positivity for Ki67.

In a study conducted by Wilson TC et al (2015)(87), Ki67 expression aids

in differentiation between basal cell adenoma and its malignant counterpart.

While histological and immunohistochemical differensiation using myoepithelial

markers proves to be difficult to differentiate basal cell adenoma and basal cell

73

adenocarcinoma, increased Ki67 expression (>5%) is seen in basal cell

adenocarcinoma, when compared basal cell adenoma can be useful in

distinguishing between the two entities.

In case of malignant neoplasms, 5 out of 11 showed Ki67 positivity. A

total of 7 mucoepidermoid carcinomas were stained for Ki67 out of which 3

were positive. According to a study conducted by Skalova et al(1994)(88), in

which 46 cases of mucoepidermoid carcinoma were studied, low Ki67

expression had a benign clinical course whereas high Ki67 expression was

associated with aggressive clinical behaviour.

We had 2 cases of acinic cell carcinoms in our study and both were

negative for Ki67 expression. In a study conducted by Hellquist et all (1997)(89),

in 32 acinic cell carcinomas they conclude that ki67 is a significant marker of

acinic cell carcinoma and is also an independant prognostic factor for survival

of patients of acinic cell carcinoma.

Other malignant tumors in our study including one case of malignant

oncocytoma and one case of salivary duct carcinoma, were both positive for

Ki67 expression.

P63 is a member of p53 family of transcription factors. It is essential for

normal epithelial cell survival. p63 is expressed in nuclei of myoepithelial and

basal duct cells in normal salivary glands. In a study conducted by Bilal H et al

(2003)(90), p63 was seen to be expressed by all salivary gland tumors that

differentiated towards luminal and myoepithelial lineages.

74

In accordance to the above mentioned study, all the benign tumors in our

study including 16 cases of pleomorphic adenoma, 2 cases of Warthin’s and one

case of basal cell adenoma were all positive for p63 expression.

In Warthins tumour, the basal cells were seen to express p63 positivity, in

accordance to the study conducted by Bilal H et al loc.cit (2003) , according to

which p63 is expressed by the basal cells of both Warthins tumour and

oncocytoma.

In our study which constituted of 11 malignant cases, except for 2 cases

of low grade mucoepidermoid carcinoma all other malignant tumors were

negative for p63 immunostaining. According to the study conducted by

Nermine M Abd Raboh et all (2015)(77), the study states that there was no

difference in the staining pattern of p63 in different tumour grades of

mucoepidermoid carcinoma.

p63 staining was found to be negative in all other malignant salivary

gland neoplasms. In the study conducted by Sams RN et al (2013)(91), it is said

that differentiation of acinic cell carcinomas from mucoepidermoid carcinoma

can be histologically difficult as both may exhibit mucin production and in such

cases p63 staining helps to differentiate the two entities.

75

SUMMARY

The P value calculated for association between malignant salivary gland

tumours and ki67 expression was found to be statistically significant.

The association between benign salivary gland tumours and p63

expression was statistically significant.

The P value calculated for the association between Pleomorphic

adenoma and Ki67 expression was found to be statistically significant.

76

CONCLUSION

From the above study we conclude

- Ki67 expression was seen significantly more in malignant salivary gland

tumours when compared with the benign tumours.

- p63 expression was seen to show significantly higher expression in

benign tumors of salivary glands.

- Ki67 was expressed in 4 cases of pleomorphic adenoma implying an

increase in tendency for recurrence or underlying malignancy in those

four cases , which are currently under follow up.

- Ki67 expression increases with increasing malignant potential of the

lesion

At the end of this pilot study, we conclude that the expression of Ki67

was very much significant in cases of malignant salivary neoplasms and the

expression increases with increasing grade of malignancy. p63 expression was

mostly seen in benign tumours which was statistically significant.

This is an ongoing study and evaluation of the IHC expression of the

studied markers with more number of cases could throw more light on

significance of the role of these IHC markers.

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APPENDIX -1 : CLINICAL PROFORMA

1.NAME-

2.AGE & SEX-

3.IP NUMBER-

4.CLINICAL HISTORY-

5. LOCAL EXAMINATION-

8.DIAGNOSIS

9.FNAC REPORT-

10.OTHER INVESTIGATIONS-

APPENDIX 2: CONSENT FORM

ஆரா சி தகவ தா

தி ெந ேவலி ம வ க அரெபா ம வமைன வ ேநாயாளக ஒஆரா சி நைடெப வ கிற .

ஆ தைல ைப ெபய : Ki67 ம p63 ஒஇ ேனாஹி ேடாெகமி யா ெவள பா -

உமி ந ர ப உட க கைள ப றியஆ ,

ந க இ த ஆரா சிய ப ேக க நா கவ கிேறா . கைள அ ல க கைளெவளய ேபாேதா அ ல ஆரா சிய ேபாேதாத கள ெபயைரேயா அ ல அைடயாள கைளேயாெவளயடமா ேடா எ பைத ெத வெகா கிேறா .

இ த ஆரா சிய ப ேக ப த க ைடயவ ப தி ேப தா இ கிற . ேம ந கஎ ேநர இ த ஆரா சியலி ப வா கலாஎ பைத ெத வ ெகா கிேறா .

இ த சிற ப ேசாதைனகள கைளஆரா சிய ேபா அ ல ஆரா சிய வத க அறிவ ேபா எ பைத ெத வெகா கிேறா .

ஆரா சியாள ைகெயா பப ேக பாள ைகெயா ப

ேததி:

ஆரா சிஒ த க த

ஆரா சிதைல : : Ki67 ம p63 ஒஇ ேனாஹி ேடாெகமி யா ெவள பா -

உமி ந ர ப உட க கைள ப றியஆ ,

ெபய : ேததி:

வய : உ ேநாயாளஎ :

பா : ஆரா சி ேச ைகஎ :

இ த ஆரா சிய வவர க அதேநா க க ைமயாக என ெதளவாகவள க ப ட . என வள க ப ட வஷய கைளநா ெகா என ச மத ைதெத வ கிேற .

ேநா க ம பாதி க றிஆரா சியாள ற வ வள க ெப ேற .

இத ேதைவயான உட ப ேசாதைன ,

இர த ம சி ந ப பா ச ப த ப டப ேசாதைனக மனமார ச மதி கிேற .

ைகெயா ப

S.

NO

PATH

NOAGE SEX CONDITION TYPE

SALIVARY

GLANDKi67 P63

1 591/11 32 F benign

Pleomorphic

adenoma -

myoepithelial rich

Submandibular negative strong +

2 686/11 38 F malignantmucoepidermoid

carcinomaparotid negative negative

3 1014/11 60 M malignantmucoepidermoid

carcinomaparotid negative negative

4 1052/11 14 M benignPleomorphic

adenomaSubmandibular 1+ moderate+

5 1115/11 55 F benignMonomorphic

adenomaParotid negative na

6 1133/11 31 M benignPleomorphic

adenomaSubmandibular 1+ moderate+

7 1341/11 15 F benignPleomorphic

adenomaparotid negative strong +

8 1652/11 13 M benignPleomorphic

adenomaSubmandibular negative mild +

9 1818/11 40 F benignPleomorphic

adenomaparotid negative na

10 1163/12 15 M benign

Pleomorphic

adenoma -

myoepithelial rich

Minor negative moderate+

11 1279/12 33 F benign

Pleomorphic

adenoma -

myoepithelial rich

Minor negative na

12 2166/12 55 M benign

Pleomorphic

adenoma -

myoepithelial rich

parotid negative na

13 396/13 27 F benignPleomorphic

adenomaparotid negative na

14 988/13 55 F benignPleomorphic

adenomaparotid negative na

15 1006/13 40 F benign Oncocytoma parotid 1+ na

16 2535/13 45 F benignPleomorphic

adenomaparotid negative na

17 3065/13 27 M benignPleomorphic

adenomaparotid negative na

18 394/14 32 F benignPleomorphic

adenomaparotid negative mild +

19 514/14 55 M benignPleomorphic

adenomaparotid negative moderate+

20 724/14 59 F benignPleomorphic

adenomaparotid negative na

21 994/14 45 F benignPleomorphic

adenomaSubmandibular negative moderate+

22 1316/14 47 F benignPleomorphic

adenomaparotid negative na

23 1369/14 19 M benignPleomorphic

adenomaparotid negative moderate+

24 1556/14 41 F malignantmucoepidermoid

carcinomaparotid 2+ mild +

25 1658/14 41 M benignPleomorphic

adenomaSubmandibular negative strong +

26 1988/14 38 F benignPleomorphic

adenomaSubmandibular negative na

27 2059/14 60 F benign Basal cell adenoma parotid negative mild +

28 2657/14 55 M malignantmucoepidermoid

carcinomaMinor negative negative

29 3012/14 57 M benign Warthins parotid negative mild +

30 336/15 58 F benignPleomorphic

adenomaparotid 2+ na

31 560/15 49 M malignantmucoepidermoid

carcinomaparotid 1+ mild +

32 1101/15 20 F benignPleomorphic

adenomaparotid negative na

33 1366/15 40 F benign Basal cell adenoma parotid negative mild +

34 1399/15 48 F benignPleomorphic

adenomaSubmandibular negative moderate+

35 1430/15 60 F benignPleomorphic

adenomaparotid negative moderate+

36 2186/15 26 M malignantmucoepidermoid

carcinomaparotid negative mild +

37 2546/15 26 F benignPleomorphic

adenomaSubmandibular 1+ na

38 2607/15 69 M malignantAcinic cell

carcinomaparotid negative negative

39 3284/15 60 M benign Warthins parotid negative moderate+

40 3931/15 18 M benignPleomorphic

adenomaparotid negative strong +

41 241/16 37 M benignPleomorphic

adenomaparotid negative moderate+

42 651/16 55 M malignantSalivary duct

carcinomaparotid 2+ negative

43 1028/16 55 M malignantMalignant

Oncocytomaparotid 2+ negative

44 1079/16 21 F benignPleomorphic

adenomaparotid negative na

45 1173/16 71 M malignantAcinic cell

carcinomaparotid negative negative

46 2118/16 70 M malignantmucoepidermoid

carcinomaMinor 1+ negative