Immunohistochemical Expression of Bcl-2 in Endometrial Carcinoma and Its Correlation with Hormone Receptor Status and Transforming Growth Factor

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IMMUNOHISTOCHEMICAL EXPRESSION OF BCL-2 IN ENDOMETRIAL CARCINOMA & ITS CORRELATION WITH

HORMONE RECEPTOR STATUS AND TRANSFORMING GROWTH FACTOR RECEPTOR

A thesis submitted to

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

in partial fulfillment of the requirements for the award of the degree of

M.D in PATHOLOGY

DEPARTMENT OF PATHOLOGY

PSG INSTITUTE OF MEDICAL SCIENCES & RESEARCH PEELAMEDU, COIMBATORE- 641 004

TAMILNADU, INDIA

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CERTIFICATE

This is to certify that the dissertation work entitled

“IMMUNOHISTOCHEMICAL EXPRESSION OF BCL-2 IN ENDOMETRIAL CARCINOMA & ITS CORRELATION WITH HORMONE RECEPTOR STATUS AND TRANSFORMING GROWTH FACTOR RECEPTOR ” submitted by Dr.Poongothai.P, is a work done by her during the period of study in this department from 10/07/2014 to 9/07/2017. This work was done under the guidance of Dr. S.Shanthakumari, Professor, Department of Pathology, PSG IMS&R.

Dr.S.Shanthakumari MD Dr. Prasanna N Kumar MD

Professor, Professor & HOD,

Department of Pathology, Department of Pathology,

PSG IMS & R, PSGIMS & R

Coimbatore - 04. Coimbatore – 04

Dr.S.Ramalingam MD Dean

PSGIMS & R Coimbatore – 04

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DECLARATION

I Dr.Poongothai P, do hereby declare that the thesis entitled

“IMMUNOHISTOCHEMICAL EXPRESSION OF BCL-2 IN ENDOMETRIAL CARCINOMA & ITS CORRELATION WITH HORMONE RECEPTOR STATUS AND TRANSFORMING GROWTH FACTOR RECEPTOR” is a bonafide work done by me under the guidance of Dr.S.Shanthakumari, Professor, Department of Pathology, PSG Institute of Medical Sciences & Research. This study was performed at the PSG Institute of Medical Sciences & Research, Coimbatore, under the aegis of the The Tamilnadu Dr.M.G.R Medical University, Chennai, as part of the requirement for the award of the MD degree in Pathology.

Dr. Poongothai. P

MD (Pathology) postgraduate, Department of Pathology, PSGIMS&R,

Coimbatore-04.

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ACKNOWLEDGEMENT

It gives me an immense pleasure to express my sincere gratitude and heartfelt thanks to my guide Dr.S.Shanthakumari, Professor of Pathology, PSGIMS&R for her invaluable guidance, immense patience, timely advice and constant support.

I sincerely thank Dr. Prasanna N Kumar, our beloved HOD, a truely inspiring teacher & Pathologist for her support and encouragement.

I extend my gratitude to Dr. T.M. SubbaRao, Professor of Pathology for his valuable guidance and support.

I would like to thank all my associate and assistant professors for their constant support and guidance.

I thank all my senior and junior postgraduates and my colleagues.

I am very thankful to our senior technician Mrs. Angeline Mary and other technical staff members, secretary staffs and attenders of our department for their timely help.

I am extremely greatful to my husband Dr. Suresh Ananth, my parents, sisters and in-laws for understanding me & supporting me during all these years.

Last but not least I must thank my sons Master.Adhav & Master. Adhvaidh, who missed me a lot but learnt with it and yet gave their love freely, during the paltry amount of time I could spend with them.

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Endometrial carcinoma is classified as Type I and Type II endometrial carcinoma. Type I endometrioid tumors are commonest tumors and 80% of the tumor belongs to this type. Type I tumors presents at a younger age with good prognosis as they present to us at an early stage and histologically low grade.

These tumors are hormone dependent. The occurrence of type II tumors are rare compared to type I tumors. They are high grade, aggressive in nature with poorer prognosis ⁽⁴⁾.

In the era of targeted therapy immunohistochemical markers plays an important role in the treatment and prognosis of endometrial carcinoma.

Estrogen and progesterone receptors are important prognostic markers. The presence of these receptors in endometrial carcinoma is associated with good prognosis.

Her 2/neu is a member of epidermal growth factor receptor. Her 2 neu mutation plays an important role in the pathogenesis of numerous cancers including endometrial carcinoma. Because of the invention of targeted therapy against

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mutated Her 2/neu receptors, the identification of Her 2 overexpression is essential in various tumors. Her 2/neu expression is seen in higher grade and advanced stage of endometrial carcinoma with poor survival⁽⁵⁾.

Apoptosis plays an important role in the development of cancer. Bcl-2 is a proto- oncogene which inhibits apoptosis and their dysfunction leads to malignancy. Bcl-2 expression is associated with many tumors including carcinoma breast, prostate lung and endometrium. In endometrial carcinoma it is related with good prognosis and may play a vital role in progression of growth from stage I to stage IV⁽⁶⁾.

Treatment and longevity of life depends on staging and prognostic factors like ER, PR, Her 2/Neu and Bcl-2.

Therefore we propose to study the immunohistochemical expression of Bcl-2 in endometrial carcinoma reported from our institute and its correlation with hormone receptor status and transforming growth factor.

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AIMS AND OBJECTIVES

1. To study the immunohistochemical expression of Bcl-2 in endometrial carcinoma

2. To study the correlation of Bcl-2 expression with hormone receptor status, transforming growth factor receptor and stage and grade of the tumor.

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REVIEW OF LITERATURE

EMBRYOLOGY OF UTERUS AND FALLOPIAN TUBES

The sex of an embryo depends on fertilization of ovum by X or Y chromosome containing sperm. During the fifth week gonadal ridge is formed. The gonadal ridge is the indication of development of reproductive organs (male or female).

Initially the gonadal ridge has bigonadal potential. The male embryo has SRY (sex determining region Y) and secretes testosterone which promotes the growth and differentiation of the Wolffian duct into the male genital system. It also secretes antimullerian hormone, which causes the regression of the mullerian ducts ⁽⁷⁾.

In the female embryo, the Wolffian duct degenerates, and the Mullerian duct contributes greatly to structure of the female reproductive system. The distal ends of the paired paramesonephric ducts fuse to form the vagina and uterus.

The uterine tubes are formed from the unfused proximal portions. The development of female genital tract and urinary tract occur in close proximity and simultaneously. Hence developmental anomaly of urinary system and genital tract is often associated⁽⁸⁾.

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ANATOMY OF UTERUS 1. LOCATION:

The uterus is a pelvic organ located behind the bladder and infront of the rectum. It is covered partially by peritoneum, which extends deeper in the posterior than the anterior aspect. Laterally, the peritoneum forms the broad ligament which unsheathes the fallopian tubes and the lymphatics and blood vessels of the uterus. The utero-ovarian ligament connects the ovary to the uterus. The other supports of the uterus are the cardinal ligaments, uterosacral ligaments and the pubocervical ligaments ⁽⁷⁾.

2. GROSS ANANTOMY:

The uterus is antiverted, pear-shaped, hollow muscular organ. In nulliparous women uterus measures 8x5x2.5 cm, weighs between 40-100gms⁽⁵⁾. Multigravida uterus (four or more deliveries) measures about 10x5x2.5 cm and weighs around 250gms. It is measurement vary with age, menstrual phase and parity. At birth cervix and corpus are equal in size, in adult the cervix is smaller than the corpus.

Uterine corpus divided into four parts i. Fundus

ii. Cornu iii. Body iv. Isthmus

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The portion of the corpus above the line connecting the insertion of fallopian tube is the fundus. The cornua is the two lateral portion of the fundus in association with intramural portion of the fallopian tubes. The isthmus is an annular zone, which lies between the cervix and the corpus. During late pregnancy and labour isthmus becomes the lower uterine segment.

The uterine cavity is triangular in shape and about 6 cm in length. It communicates with the lumen of the each fallopian tubes at the cornua and with the vagina through the cervical canal⁽⁹⁾.

BLOOD SUPPLY:

The uterus is supplied by right and left uterine arteries. They are divided into ascending and descending branch at the level of isthmus. The ascending and descending branch of the uterine arteries forms the arcuate arteries. These arcuate arteries are complex network of circumferentially arranged subserosal arteries. Radial arteries originating from these arteries penetrate the myometrium. These in turn give rise to the straight arteries supplying the basal region of the endometrium and the spiral arteries which supply the rest of the endometrium ⁽¹⁰⁾.

LYMPHATIC DRAINAGE:

Lymphatic channels from parametrium and paracervical region drain into the inguinal nodes & obturator nodes. From the obtruator nodes it goes to

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hypogastric nodes, the external and common iliac nodes, the sacral nodes and finally into the aortic nodes.

HISTOLOGY OF THE UTERUS:

Uterus is lined by endometrium, it provides the environment for fetal development. The thick muscle wall, the myometrium, expands during the pregnancy and serosa forms the outer layer.

THE ENDOMETRIUM:

The endometrium has variable thickness measuring between 1-5 mm at different stages of the menstrual cycle. It is composed of glands and stroma.

The glands show columnar epithelial lining. It varies in size and shape according to the phases of the menstrual cycle. The stroma is composed of elongated endometrial cells, uterine natural killer cells, lymphocytes and spiral arteries.

PHASES OF MENSTRUAL CYCLE:

Under the influence of estrogen and progesterone secreted by ovary, the endometrium undergo cyclic changes through three distinct phases. They are

Menstrual phase

Proliferative phase

Secretory phase

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MENSTRUAL PHASE ( DAY 1 TO 4):

In the absence of fertilization, degeneration of corpus luteum takes place due to cessation of progesterone and estrogen secretion. In the absence of progesterone, endometrium cannot be sustained. Spasmodic contraction in the spiral arterioles of the stratum functionalis leading to ischemia and degeneration of the superficial layer of the endometrium and leakage of blood into the stroma. Stromal cells disaggregate and the glands fragment. The coordinated collapse of the functionalis with necrotic predecidua and sheets of neutrophils is called scheduled breakdown. Further ischemia leads to degeneration of the whole stratum functionalis and they shed as menses.

Because of the local release of the anticoagulant factors the menstrual blood dose not clot normally. By the 3 to 4 th day of menstruation, proliferation of the basal endometrium has begun⁽¹¹⁾.

PROLIFERATIVE PHASE:

In this phase endometrium has been progressively increased in thickness due to proliferation of glands, stroma and vessels. During early proliferative stage, the glands are nonbranching, nonbudding similar shaped and are lined by columnar epithelium with pseudostartification of the nuclei. There is increase in mitosis both in the epithelium and in the stroma. The stroma is highly cellular in this phase. During mid and late proliferative phase the glands become tortuous and closely packed. The stroma is edematous with occasional lymphoid aggregates ⁽¹²⁾.

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EARLY SECRETORY PHASE :( POSTOVULATORY DAYS 2 TO 5 ) After the ovulation the endometrium shows secretory differentiation due to the action of progesterone. The endometrium is composed of coiled glands and are lined by cells similar to that present in the proliferative phase and most of the cells show vacuolation in their cytoplasm. The cytoplasmic vaculoation is due to the glycogen which is synthesized by glandular epithelium under the influence of progesterone.

MIDSECRETORY ENDOMETRIUM: (Postovulatory Days 6 To 8)

The secretory material appears in the lumen by day 19.The peak of intra glandular secretions is seen at day 21 with coiling of spiral arterioles and edema of the stroma⁽⁵⁾.The absence of vaculoation and predecidua is the most useful indicator of midsecetory phase. This is a time of implantation of the free blastocyst if fertilization has been taken place in this cycle⁽¹²⁾.

LATE SECERTORY ENDOMETRIUM: (Postovulatory Days 9 To 14) Predecidual transformation of stromal cell begins around the spiral arterioles on tenth postovulation day and later involves the stroma. These changes make spiral arteries predominant in the low power view in the beginning of this phase. At the end of late secretory phase, the predecidual stromal reaction under the surface epithelium coalesces with predicidual cells around the arterioles forming large sheets of predecidua. Finally stromal granulocytes appear and are more prominent.

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ARIAS- STELLA REACTION:

The endometrium is composed of coiled glands, with vacuolations of lining cells. The cells are large with abundant clear to eosinophilic cytoplasm with pleomorphic hyperchromatic nuclei Stroma shows decidual changes. These changes are due to the effect of increased gonadotrophin and seen in normal pregnancy, gestational trophoblastic disease, ectopic gestation and hormone therapy ⁽³⁾.

THE ATROPHIC ENDOMETRIUM:

After the menopause, endometrium is thin consisting only of stratum basalis with sparse and inactive glands. The glands may become cystically dialated and are lined by cuboidal to low columnar epithelium. The stroma is less cellular and contains more collagen fibres with no mitotic activity⁽¹³⁾.

MYOMETRIUM:

Myometrium is the main bulk of the uterus, which consists of smooth muscle cells and is composed of interlacing bundles of long slender fibers. These fibers are arranged in transverse, longitudinal and oblique sections. There is rich network of arteries, veins and collagen fibers within the muscle fibers. During pregnancy the uterus increases in size due to hypertrophy as well as hyperplasia of the uterine muscle fibers⁽⁷⁾.

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DISEASES OF THE UTERUS:

Diseases of the uterus can be classified as

 Inflammatory disorders

Inflammation of the endometrium and myometrium are uncommon because of endocervical barrier action. Acute endometritis is usually associated with retained product of conception, after delivery or miscarriage and due to staphylococci , hemolytic streptococci and other bacteria. Chronic endometritis is due to tuberculosis, Chalmydia and are associated with pelvic inflammatory disease and intrauterine contraceptive devices ⁽¹⁴⁾.

 Precancerous condition:

Endometrial hyperplasia is a common precancerous lesion for endometrial carcinoma. It can be classified as simple and complex hyperplasia. Both are again classified as simple hyperplasia with atypia and without atypia, complex hyperplasia with atypia and complex hyperplasia without atypia.

 Benign and malignant neoplasm:

Endometrial carcinomas are common than than malignant mesenchymal neoplasm of the uterine corpus.

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ENDOMETRIAL CARCINOMA

Endometrial carcinoma is the commonest malignancy occurring in women of developed and industrialized countries. It accounting for 7% of female cancers.

Postmenopausal women are mostly affected than premenopausal women with median age group of 60 years⁽¹⁵⁾. 2-14% of cancers arise in patients younger than 40 years ⁽¹⁶⁾. Endometrial adenocarcinoma has been classified into Type I (Endometrioid) & Type II (Non Endometrioid). 80% to85% of cases belongto Type I Endometrioid adenocarcinoma. The 10% to 15% of cases belong to Type II ⁽⁴⁾. Type I tumors usually occurs in younger, perimenopausal women, with longer history and is related with unopposed estrogen. This neoplasm is low grade with good differentiation and predominantly endometrioid type.

Type II endometrial carcinomas usually occurs in older women with short history, higher grade tumors and are mainly of serous or clear cell carcinoma type ⁽⁵⁾. These tumors are not related to hormones and has a poorer prognosis.

Type I tumors usually arise in the background of endometrial hyperplasia whereas type II tumors are associated with atrophic endometrium.

PATHOGENESIS:

Unopposed estrogens is the main cause for the type I carcinoma. Estrogens in hormone replacement therapy is a influencing factor in older females. These patients are commonly obese, diabetic, hypertensive, nulliparous or have late menopause. These tumors are associated with endometrial hyperplasia specifically with atypical hyperplasia. Non- endometrioid adenocarcinomas

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usually seen in the seventh and eighth decades and is not related with excess estrogen, diabetes or obesity⁽¹⁶⁾.

WHO histological classification of endometrial carcinoma Endometrioid adenocarcinoma

Variant with squamous differentiation

Villoglandular variant Secretory variant

Ciliated cell variant

Mucinous adenocarcinoma

Serous adenocarcinoma

Clear cell adenocarcinoma

Mixed cell adenocarcinoma

Squamous cell carcinoma Transitional cell carcinoma

Small cell carcinoma

Undifferentiated carcinoma⁽¹⁷⁾.

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RISK FACTORS FOR ENDOMETRIAL CARCINOMA Estrogens

Excessive estrogen is usually related with increased risk of endometrioid adenocarcinoma. It may be endogenous or exogenous.

Exogenous estrogen stimulation is seen in postmenopausal women who have taken estrogen and progesterone hormones. The risk of developing endometrial carcinoma increases while taking estrogen alone and decreases with additional progesterone therapy. The risk for the development of endometrial carcinoma persists even for several years after discontinuation of estrogens.

Tamoxifen is an antiestrogen, which is used as adjuvant therapy for patient suffering from carcinoma breast with positive estrogen receptor. The drug has dual effect on the endometrium. In childbearing age group women these drug has antiestrogenic effect, but in women in postmenopausal age it has a weak estrogenic effect with increased risk of endometrial carcinoma⁽¹⁸⁾. Also the risk of endometrial carcinoma in patient on Tamoxifen therapy is related to the longer duration of the treatment and preexisting lesions of the endometrium such as polyp and hyperplasia⁽¹⁹⁾.

Early menarche, late menopause, anovlatory cycles, low parity and infertility are associated with increase lifetime endogenous estrogen exposure. This is also a risk factor for developing endometrial carcinoma⁽³⁾.

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Some of the ovarian disease producing excess estrogen and cause endometrial hyperplasia and endometrial carcinomas. These diseases can be neoplastic or non neoplastic. Granulosa cell tumors is a sex cord stromal tumor, that usually affect the postmenopausal women with increased production of estrogens.

Endometrial carcinoma arises in 9-12% of women with this tumor ⁽²⁰⁾. Thecomas also produces excess estrogen, it occurs in women over the age of 40 years. This increased estrogen production leads to endometrial hyperplasia and carcinoma.

In non neoplastic ovarian diseases poly cystic ovarian disease (PCOD) and hyperthecosis produces excess estrogen and androgens. PCOD occurs in younger patients, the carcinoma of endometrium arises less than 5% of women with PCOS disease⁽²¹⁾ and hyperthecosis may occur in peri and postmenopausal women.

Other risk factors are obesity, diabetes, Hereditary Non Polyposis Colonic Cancer syndrome (HNPCC). Lynch syndrome (Hereditary nonpolyposis cancer syndrome [HNPCC]) is due to germline mutation in mismatch repair genes.

Endometrial cancer occur earlier in women with Lynch syndrome than in women with sporadic endometrial cancers ⁽¹²⁾.

Increased physical activity, coffee and smoking have been revealed to have a protective effect. Cigarette smoking reduces the risk of endometrial carcinoma primarily in postmenopausal women ⁽²²⁾.

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PRECURSOR LESION:

Endometrial hyperplasia is a precursor lesion of endometrial carcinoma. In endometrium the cell proliferation and cell death are dependent on hormone status. The development of endometrial carcinoma is a multistep process, this includes simple hyperplasia, complex endometrial hyperplasia without cytological atypia and complex hyperplasia with atypia also called as atypical endometrial hyperplasia, finally lead to endometrial carcinoma ⁽²³⁾.

Most of the endometrioid adenocarcinoma are preceded by endometrial hyperplasia. Proliferation of endometrium is estrogen dependent. Continuous unopposed estrogen stimulation leads to disordered proliferation and then simple hyperplasia. Increased gland to stromal ratio is a characteristic finding in all the forms of endometrial hyperplasia, the ratio is more in complex hyperplasia. The risk of development of carcinoma is more from complex hyperplasia with atypia (atypical endometrial hyperplasia) than complex hyperplasia without atypia⁽²³⁾.

 Microsatellite instability is present in atypical hyperplasia and not in hyperplasia without atypia⁽²⁴⁾.

 Simple hyperplasia and complex hyperplasia without atypia are polyclonal lesions where as atypical hyperplasia and endometrial carcinoma are monoclonal lesions ⁽²⁵⁾.

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The atypical hyperplasia has extreme glandular crowding and little intervening stroma, closely resembling carcinoma. Adenocarcinoma are diagnosed by presence of complex and confluent glandular pattern, an extensive papillary pattern and stromal infiltration of glands with desmoplastic response⁽¹²⁾.

PRECURSOR LESION FOR SEROUS CARCINOMA:

Non endometrioid carcinoma has different molecular pathogenesis. Serous carcinomas develop from the normal endometrium due to p53 mutation. p53 mutation leads to accumulation of p53 protein in normal cells nuclei and this premalignant condition called as p53 signature. The endometrial glandular dysplasia leads to serous endometrial intraepithelial carcinoma ⁽²⁶⁾.

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FIGURE 1:Relations among proliferation, hyperplasia, atypical hyperplasia and endometrial carcinoma

Postmenopausal persistent proliferation Premenopausal

proliferative endometrium

DISORDERED PROLIFERATION

COMPLEX HYPERPLASIA SIMPLE HYPERPLASIA

?spontaneous origin not preceded by

ATYPICAL HYPERPLASIA (Complex & Simple)

?spontaneous origin not preceded by

ADENO CARCINOMA

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MOLECULAR GENETICS:

Endometrioid adenocarcinoma and serous carcinoma have a different molecular pathogenesis. Mutation in the PTEN gene is responsible for 83% of endometrioid carcinoma ⁽²⁷⁾. PTEN is usually expressed in glands during the normal proliferative phase which are estrogen driven and also in endometrial hyperplasia. For development of cancer, the cell should undergo one initiating mutation and several additional mutation. PTEN is a tumor suppressor gene located in the chromosome 10q23⁽¹⁴⁾ and it inhibits the action of phosphatidylinosito l3-kinase (PI3K) on phosphatidylinositol–3,4,5- triphosphate (PIP3) and preventing the progression of PI3/AKT pathway.

PTEN get mutated through deletion, point mutations or epigenetic silencing.

PTEN mutation lead to loss of function this gene lead to continued progression of PI3/AKT pathway and continued proliferation ⁽²⁸⁾. In type I endometrioid carcinoma PTEN is the initiating mutation and the other mutations includes K- RAS mutation, beta catenin inactivation, loss of cyclin D1 activity, microstatellite instability and BCL 2 over expression⁽²⁹⁾. (Figure 2)

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GLANDULAR EPITHELIUM

ALTERED RESPONSE TO HORMONES

CLONAL SELECTION

EIN

ENDOMETROID CARCINOMA

PAX 2 DOWN REGULATION PTEN MUTATION

UNOPPOSED ESTROGEN

ADDITIONAL MUTATIONS K RAS

ADDITIONAL MUTATIONS KRAS, BETA CATENIN, OTHERS

Figure 2

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More than 80% serous carcinomas are due to mutation in p53 tumor suppressor gene. p53 is responsible for programmed cell death in DNA damaged cells . p53 overexpression has been found more commonly in non endometrioid carcinomas and is also seen in high grade type I tumors. Mutation in p16, reduced expression of E-cadherin and over expression of human epidermal growth factor receptor 2 (Her-2/neu) are also responsible for the development of non-endometrioid carcinomas⁽³⁰⁾.

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Figure 3

AGING PROCESS, REPAIR

SURFACE EPITHELIUM

GENOMIC DAMAGE

P53 MUTATION

ENDOMETRIAL GLANDULAR

DYSPLASIA

PAX 2

DOWNREGULATION

INVOLUTION SEROUS EIC

INVASION

UPSC

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ENDOMETRIOID ADENOCARCINOMA

This is a type I endometrial carcinoma of endometrium characterized by a glandular pattern and the glands are similar to the proliferative endometrium.

Clinical features:

These tumors predominantly occurs in sixth and seventh decades. Most of these tumors develops slowly in the hyperestrogenic state along with endometrial hyperplasia. The endometrial carcinoma patients usually present with irregular bleeding and discharge. Some patients are asymptomaticc and are diagnosed by endometrial curetting which is done before the initiation of hormone replacement therapy ⁽³⁾. In advanced cases with invasion pelvic pain, pressure symptoms and metastasis related symptoms are seen⁽⁹⁾.

The uterus may be grossly or faintly enlarged. In some patients uterus may be normal in size or atrophic especially in postmenopausal women. The tumors usually arises in the body of the uterus, but can arise in lower uterine segment especially in younger females⁽⁹⁾. It may present as a single dominant mass or two to three separate masses or as polyps or as diffuse thickening of the endometrium. The mass is exophytic and irregular. Often the surface shows ulceration. It may invade the myometrium superficially or deeply. In advanced cases the mass infiltrate beyond the serosa and into the cervix.

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Microscopically these tumors are composed of glands similar to normal proliferative endometrium. The glands are arranged in villoglandular pattern, solid sheets and cribriform pattern. The cells are large with round nuclei, clumped chromatin and have prominent nucleoli. The lumen of the glands sometimes filled with cell debris and is known as dirty necrosis, usually related with high grade tumors, myometrial invasion and extension of the tumor into the isthmus. The stroma shows desmoplasia and few foamy cells. Desmoplasia may be absent in diffusely infiltrating tumor⁽³¹⁾. These foamy cells in the stroma are due to reactive changes because of tumor cells death.

Criteria for malignancy

 Confluent cribriform growth

 Villoglandular architecture

 Conspicuous nuclearatypia

 Solid areas

VARIANTS :

 With squamous differentiation:

Squamous component is presents almost one fourth to half of the endometrioid adenocarcinoma. It may be due to metaplasia or neoplasia. When the squamous component is well differentiated it is called adenoacanthoma. Adenosqumaous is named when the squamous component is poorly differentiated.

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Criteria for squamous differentiation:

1. Keratin and keratin pearls

2. Intercelluar bridges

3. Three or more of the following criteria

- sheet like growth without gland formation with distinct cell margins

-eosinophilic and thick/ glossy cytoplasm

-A decreased nuclear to cytoplasmic ratio compared with the rest of the tumor⁽¹⁷⁾.

 Villoglandular variant

This variant exhibit numerous well defined papillae with discrete fibrovascular core and lined by pseudostratified endometrioid type epithelium. The nuclei usually have grade I features. These variants are usually associated with good prognosis⁽³⁾. But invasive villoglandular papillary pattern is associated with poor prognosis.

 Secretory variant

This is a rare variant of endometrioid adenocarcinoma. In this variant the neoplasm are usually composed of glands lined by cells with basal and supra nuclear vacuoles and resembles early and mid secretory endometrium.. These changes may be due to progestin therapy or a feature of the malignant cells. In

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Clear cell carcinoma the cells are cuboidal with clear cytoplasm and exhibit significant nuclear atypia. Secretory adenocarcinomas are related with good prognosis⁽³²⁾.

 Tubal ( ciliated cell ) variant:

It is a rare variant. Occasional ciliated cells may be seen in most of the cases.

If only 75% of the glands lined by ciliated cells, the tumor is termed as ciliated cell carcinoma. It has a favorable prognosis.

 Endometrioid carcinoma with sertoliform differentiation:

In this variant endometrioid carcinoma have the area composed of glands arranged in closely packed tubules or trabeculae and resembling sex cord stromal tumors. The glands are lined by cells with clear cytoplasm and basely located nuceli⁽⁹⁾.

 Oxyphilic cell carcinoma:

This is a rare variant of endometrioid adenocarcinoma and composed of large cells with abundant eosinophilic cytoplasm⁽³³⁾.

MUCINOUS ADENOCARCINOMA:

Mucinous adenocarcinoma accounts up to 9% of endometrial carcinoma. It is frequently associated with an endometrioid component. In mucinous adenocarcinoma atleast 50% of tumour cells show intra cytoplasmic mucin.

Mucinous adenocarcinoma is more common in women who received tamoxifen

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and progesterone therapy⁽³⁴⁾. The distinction between mucinous adenocarcinoma of the endometrium and endocervix can be very difficult particularly in a biopsy or curettage specimen. The prognosis is usually good.

PAPPILARY SEROUS CARCINOMA:

These are type II endometrial carcinoma with aggressive nature and frequently presents at an advanced stage. It is usually associated with synchronous involvement of ovary, fallopian tube and peritoneum. Usually the patient are elder than type I patients, and are not associated with excessive estrogen.

Serous intra epithelial carcinoma and endometrial glandular dysplasia have been identified as its precursor lesions. These tumors show complex papillary pattern with irregular thick papillae with cellular budding. It may also show solid growth pattern and silt like irregular spaces. The lining cells have abundant eosinophilic cytoplasm, marked pleomorphism , high N:C ratio and prominent nucleoli. Psammoma bodies are present upto 30% of the tumors.

These tumors are usually associated with myometrial invasion and lymphovascular permeation. Cervix and lower uterine segment are commonly involved in serous adenocarcinoma. Relapses are more common in these tumors.

Serous adenocarcinoma may coexist with endometrioid or clear cell adenocarcinoma and these tumors also have aggressive behavior and poor prognosis⁽³⁵⁾.

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CLEAR CELL ADENOCARCINOMA:

These tumors accounts for 5% of all endometrial adenocarcinomas. These tumors show various arrangements include tubulocystic, papillary , glandular and solid which are frequently admixed. The papillary pattern is the most common, the papillae are small and rounded. The lining cells exhibit clear cytoplasm , some of the cells have scant cytoplasm and large nuclei protruding into the lumen in a hobnail appearance. Clear cytoplasm is due to the presence of glycogen and it is most prominent diagnostic feature of this neoplasm. These tumor has marked nuclear atypia and variable mitotic figures. It has very poor prognosis⁽⁴⁾.

SQUAMOUS CELL CARCINOMA:

These tumors are occur at an older age group. Icthyosis uteri, a condition in which there is extensive squamous metaplasia of the endometrium, is considered to be its precursor. It resembles squamous cell carcinomas of other organs and has little or no adenocarcinomatous elements. In this patients cervix should be evaluated to rule out primary origin of this tumor. The prognosis very poor⁽¹⁵⁾.

MIXED ADENOCARCINOMA:

These tumors are composed of both type I and type II endometrial adenocarcinomas. The minor component should comprise at least 10% of the tumors. The prognosis is depend on the aggressive component of the tumor,

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even the presence of 25 % or more high grade is associated with poor prognosis⁽⁵⁾.

TRANSITIONAL CELL CARCINOMA:

This is also a rare tumor of endometrium with mullerian profile. The morphology of the tumor looks like urothelial carcinoma of the urinary tract.

For the diagnosis of transitional cell carcinoma the tumor should be composed of more than 90% of transitional cell. This tumor is usually admixed with squamous cell carcinoma or endometrioid or serous carcinoma⁽³⁶⁾. Because of their mullerian derivation, these tumors are positive for CK 7 and negative for CK 20⁽¹⁵⁾. Human papilloma virus type 16 has been linked to this tumor⁽⁵⁾.

SMALL CELL CARCINOMA :

These are also known as neuroendocrine carcinomas . This is very rare tumors with an incidence of less than 1% of all endometrial cancers. This tumor is similar to small cell carcinoma of the lung and usually associated with a component of typical endometrioid adenocarcinoma. This neoplasm is composed of small oval to spindle cells with scant cytoplasm, nuclei with salt and pepper chromatin. The cells are arranged in insular or trabecular growth.

Necrosis and lymphovascular invasion are common. Immunohistochemicaly these tumors are positive for cytokeratin and neuroendocrine markers. It has good prognosis ⁽³⁷⁾.

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UNDIFFERENTIATED CARCINOMA:

These tumors are identified as carcinoma but further subclassification could not be done due to lack of differentiation. The tumor has diffuse arrangements by large anaplastic cells with minimal glandular pattern. ⁽³⁾. Scant to absent stroma with focal myxoid changes are seen. These tumors show 10% of epithelial marker positivity and less than 10% cells are positive for neuroendocrine markers. These tumors has poor prognosis because more than half of the patients are diagnosed with metastasis⁽⁴⁾.

GAINT CELL ADENOCARCINOMA:

This is a rare endometrial carcinoma with high grade histology. The tumor containing sheets and nests of bizarre multinucleated gaint cells. Endometrioid or clear cell carcinoma can be accompanied with this tumor⁽³⁸⁾.

ENDOMETRIAL CARCINOMA WITH TROPHOBLASTIC DIFFERENTIATION:

These tumor has trophoblastic and nontrophoblastic areas. Synctiotrophoblast like multinucleated cells occupy the trophoblastic areas and serous carcinomas usually present in the nontrophoblasic areas. These tumors should be differentiated from gestational choriocarcinoma. These synctiotrophoblast like cells are strongly positive for human chorionic gonadotropin ( hCG) ⁽⁹⁾.

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HEPATOID ADENOCARCINOMA:

This rare tumor is usually related with high alpha - fetoprotein production and more common in postmenopausal women The tumor is arranged in trabecular pattern. The cells are polygonal with rich eosinophilic cytoplasm. Because of this morphology this tumor resembles hepatocellular carcinoma and show positive immunostaining for alpha- fetoprotein ⁽³⁹⁾.

SIGNET RING CELL ADENOCARCINOMA:

This tumor is comprising of cells with abundant clear cytoplasm and peripherally pushed nuclei. Stromal histiocytes and vacuolated decidual cells may mimic signet ring cells. The metastasis from carcinoma breast or stomach should be ruled out before make this diagnosis⁽⁹⁾.

LYMPHOEPITHELIOMA – LIKE CARCINOMA:

This a rare tumor of endometrium which have a histology similar to lymphoepithelioma like carcinoma of the nasopharnyx. This tumor is composed of nests and sheets of large cells with round nuclei with prominent nucleoli. The stroma shows dense lymphocytic infiltration. This tumor can be occur anywhere in female genital tract. Ebstein - barr is virus usually associated with lymphoepithelioma like carcinoma of cervix than endometrium. The tumor is positive for cytokeratin, EBV latent membrane protein 1 and negative for leucocyte common antigen ⁽⁴⁰⁾.

(41)

LARGE-CELL NEUROENDOCRINE CARCINOMA:

This rare tumor commonly affect postmenopausal women. The cells are arranged in solid or trabecular pattern. The cells have abundant basophilic cytoplasm with vesicular to hyperchromatic nuclei. It may coexist with type I tumors⁽⁴⁾.

Various factors play role in the prognosis of endometrial carcinoma and is as follows

FACTORS THAT IMPACT THE PROGNOSIS IN ENDOMETRIAL CARCINOMA:

Prognostic factors are useful to find the possible disease outcome and also to decide the necessity for adjuvant therapy.

The important prognostic factors are 1. Patient age and race

2. Tumor stage ( FIGO)

3. Histological type

4. Histologic grade 5. Myometrial invasion

6. Cervical extension

7. Adenexal involvement

(42)

8. Lymphovasular invasion

9. Lymphnode involvement 10. Peritoneal cytology

11. Estrogen dependence

12. Hormone receptor status

13. P53 overexpression

14. Her2 /neu expression

15. DNA ploidy

16. Cell proliferation 17. Antiapoptic marker

18. Angiogenesis

1. AGE AND RACE:

Older women have poor prognosis than younger women. Endometrial carcinoma is more prevalent in white than black women, but it has worse prognosis in black women. Serous or clear cell carcinoma most commonly occurs in black women. Poor prognosis in black people is usually due to occurrence of unfavourable type, advanced stage at diagnosis, socioeconomic factors and comorbidities⁽¹⁵⁾.

(43)

2. FIGO STAGING OF ENDOMETRIAL ADENOCARCINOMA:

The staging system for uterine cancer is first approved by the International Federation of Gynecology and Obstetrics ( FIGO) in 1958. FIGO revised its staging system subsequently .Recent revised FIGO staging for uterine cancer is published in the International journal of Obstetrics and Gynecology in the year of 2009⁽⁴¹⁾. Many studies show the stage of the tumor at the time of diagnosis is the single most important prognostic factor⁽¹⁵⁾. The patient with stage I tumors has 5 year disease free survival about 90%. The survival rate decreases with advanced stage.

(44)

Table 1: Revised FIGO staging of endometrial carcinoma

STAGE CHARACTERISTICS

I A Tumor involving the endometrium with no or < ½ of myometrium involved

I B Tumor involving the endometrium with > ½ of myometrium involved

II Cervical stromal involvement

III A Serosa of the uterus or adnexae involved III B Vaginal or parametrial involvement III C Metastasis to pelvic or para-aortic nodes

C1 - pelvic nodes involved

C2 –para –aortic nodes involved with or without pelvic node involvement

IV A Invasion of bladder and /or bowel mucosa

IV B Distant metastasis, including intra- abdominal and / or inguinal nodes

(45)

3. HISTOLOGIC TYPE:

Type I endometrioid carcinoma, its variants (squamous differentiation, villoglandular, secretory and ciliated) and mucinous adenocarcinoma are associated with good prognosis. Type II non- endometrioid adenocarcinoma which include serous adenocarcinoma, clear cell adenocarcinoma , squamous cell carcinoma and undifferentiated adenocarcinomas are associated with unfavorable prognosis. The prognosis is worst in patients with mixed tumors which contain more than 25 % of a serous, clear or undifferentiated component⁽³⁾.

4. HISTOLOGIC GRADING:

FIGO grading system for endometrioid carcinoma is based on the tumor architecture (Table 2) and nuclear morphology (Table 3). Squamous morules/

differentiation are omitted from the grading⁽¹⁷⁾. 50% of the edometrioid carcinomas are well differentiated or grade I tumors⁽⁹⁾.

Table 2: Grading of endometrial carcinoma based on tumor architecture:

GRADE CHARACTERISTICS

Grade I </= 5% tumor growth is in solid sheets

Grade II 6-50% tumor growth is in solid sheets

Grade III >50% tumor growth is in solid tumors

(46)

Table 3:Grading of endometrial carcinoma based on nuclear features

GRADE CHARACTERISTICS

Grade I Round to oval nuclei with evenly distributed chromatin and inconspicuous nucleoli

Grade II Irregular oval nuclei with chromatin clumping and moderate sized nucleoli

Grade III Large pleomorphic nuclei with coarse chromatin and large irregular nucleoli

When the nuclear grade is high , the architectural grade of grade I and II lesions is raised by one for endometrioid adenocarcinoma. For grading the non- endometrioid carcinoma only nuclear features are used , but in mucinous carcinoma architectural grading is used⁽⁴²⁾. The patients with grade I tumors has 94% of 5 year survival rate when compared to 72% of 5 year survival rate in grade III tumors ⁽¹⁵⁾.

5. MYOMETRIAL INVASION:

Myometrial invasion is an independent factor for predicting the outcome of early-stage tumors. Increasing depth of myometrial invasion associated with increasing risk of extrauterine extension, lymph node metastasis, recurrence and with decreased survival⁽⁴³⁾.

(47)

6. CERVICAL EXTENSION:

Extension of tumor into the cervix may be due to direct extension or lymphatic spread. Fractional dilatation and curettage results in 5% implantation of endometrial carcinoma of the denuded endocervix. Cervical extension is associated with high recurrence and poor survival⁽¹⁵⁾.

7. ADENEXAL INVOLVEMENT:

Assessing the spread of tumor to the adenexal structures is difficult. 5% of patient with endometrial cancer have synchronized ovarian carcinoma. The prognosis depends on type of tumor, endometrioid histology in both uterus and ovary has good prognosis with 100% survival rate⁽⁴²⁾.

8. LYMPHOVASULAR INVASION:

Invasion into vascular or lymphatic spaces is identified by the presence of nests of tumor cells surrounded by flattened endothelial cells. Presences of lymphovascular invasion is associated with increased lymphnode metastasis, high recurrence rate and poor survival⁽⁴⁴⁾. Lymphovasular invasion is more common in serous tumors than in endometrioid tumors.

9. LYMPHNODE INVOLVEMENT:

Patients with clinical stage I with lymphnode metastasis is depend on the depth of myometrial invasion. Positive para- aortic nodes are associated with worst outcome than positive pelvic nodes ⁽¹⁶⁾.

(48)

10. PERITONEAL CYTOLOGY:

Peritoneal washing shows malignant cells in 17 % of stage I and 85 % in stage IV tumors. Recurrence are more common in patients with positive peritoneal cytology⁽⁴⁴⁾.

11. ESTROGEN DEPENDENCE:

The tumor occurring in patients with Poly cystic ovarian disease and functional ovarian tumors have better prognosis. Endometrioid type tumors have good prognosis because of association with excess estrogen⁽⁹⁾.

12. HORMONE RECEPTOR STATUS:

Positive estrogen and progesterone receptors are associated with early stage, well differentiated tumors. These receptors have direct correlation with prognosis and recurrence ⁽⁴⁵⁾. Progesterone positive tumors respond well to hormonal treatment.

13. p53 OVER EXPRESSION:

p53 over expression has been found more commonly in advanced type I tumors, type II tumors and high grade tumors . p53 over expression is directly associated with unfavorable prognosis ⁽⁴⁶⁾.

(49)

14. Her 2 / NEU EXPRESSION:

Her 2/ neu over expression is an independent and negative prognostic marker for endometrial carcinoma. Her 2/ neu expression is associated with advanced stage and high grade tumor with lesser survival⁽⁴⁷⁾.

15. DNA PLOIDY:

DNA ploidy is also a relatively useful prognostic factor. Diploidy is usually associated with higher disease free survival in stage I endometrial carcinomas.

High grade and higher stage tumors are aneuploidy tumors and they have poor prognosis⁽¹⁵⁾.

16. CELL PROLIFERATION:

The tumor which have high proliferating activity associated with aggressive disease and lesser survival ⁽⁹⁾.

17. ANTIAPOPTIC MARKER:

Bcl-2 is an antiapoptotic gene. Bcl-2 expression is seen in early stage and well differentiated tumors and its presence associated with good prognosis. Loss of bcl-2 expression associated with high grade, advanced stage and poorer prognosis ⁽⁴⁸⁾.

(50)

18.ANGIOGENESIS:

Angiogenesis is important for tumor proliferation. Vascular endothelial growth factor (VEGF) expression is associated with advanced stage, high grade, lymphatic invasion and poor survival ⁽⁴⁹⁾.

IMMUNOHISTOCHEMISTRY IN ENDOMETRIAL CARCINOMA:

Immunohistochemical markers are used in endometrial carcinoma mainly for assessing the prognosis and treatment of the patient. Many studies show various markers are used in endometrial carcinoma which include estrogen, progesterone receptors, Bcl-2, Her 2 neu, BAX, p53 , Cox 2 , PTEN, Ki 67 and Cathepsin D etc⁽⁵⁰⁾.

ESTROGEN AND PROGESTERONE RECEPTOR:

The uterine endometrium is sensitive to estrogen and progesterone hormones.

These hormones regulate the growth, development and restoration of reproductive tissues. Estrogen and progesterone receptors belongs to steroid- thyroid receptor super family. These receptors are expressed in both glandular epithelium and the stroma, which is necessary for the growth and differentiation of glands and stroma. These hormones binds to the nuclear receptor protein and activate the transcription factors. Two forms of ER receptors are present including ER-α & ER-β. Both forms are encoded by different genes. Like ER , PR has A and B isoforms but both forms are encoded by same gene ⁽⁵¹⁾. ER and PR expression is more in the proliferative phase and

(51)

gradually down-regulated during the secretory phase of the menstrual cycle. In atypical endometrial hyperplasia progesterone receptor levels are lower than the normal endometrium but higher than invasive carcinoma^{(52)( 53)}.

Evaluation of ER and PR status was performed according to the method described by Carcangiu et al ⁽⁵⁴⁾( Table 5 )

The ER, PR expression in endometrial carcinoma is ranging from 32-77% &

54-72% for ER and PR respectively⁽⁴⁵⁾ . Numerous studies support the finding that high ER and PR levels are associated with a well differentiated tumor.

High ER and PR are associated with good prognosis. Low PR level is predictive of lymph node metastasis. In endometrial carcinoma surgery is main modality of treatment. The role of adjuvant therapies are radiation therapy and hormonal therapy. This hormonal therapy has more important role in the treatment for advanced and recurrent diseases. The response of hormonal therapy mainly depends on hormone receptor status especially progesterone receptor levels. Ehrlich et al demonstrated a reduction in PR associated with poor response of the patient with progestin treatment ⁽⁵⁵⁾. The survival of the patients with endometrial carcinoma mainly depends on progesterone receptor levels.

(52)

BCL-2:

Apoptosis plays an important role in carcinogenesis. Apoptotic and antiapoptotic genes produce proteins that regulate apoptosis. The dysfunction of their expression may lead malignancy. Bcl-2 gene is located on chromosome 18q21 and encode a protein , that is an important regulator of programmed cell death or apoptosis. This protein is located in the mitochondrial membrane, endoplasmic reticulum and nuclear membrane⁽⁶⁾. Now bcl-2 is associated with many tumors including carcinoma breast, prostate and lung ⁽⁵⁶⁾.

In endometrium bcl-2 is expression is seen mostly in the follicular phase.

Secretory phase shows minimal expression. Many studies support that bcl-2 is expressed high levels in simple hyperplasia ⁽⁵⁷⁾. The expression of bcl-2 is low in complex hyperplasia with atypia and invasive adenocarcinoma.

Based on study conducted by G.Guset et al, an arbitrary scoring system was drawn up for quantifying the expression of Bcl-2⁽⁶⁾. Bcl-2 expression is ranging from 25% to 85% in endometrial carcinoma ⁽⁵⁸⁾ .

Bcl-2 positivity has been associated with a favorable clinical outcome and good histopathologic factors like grade I endometrioid carcinoma and early staging.

It may play vital role in progression from stage I to stage III and IV of the tumor.

(53)

HER 2 NEU RECEPTOR:

Her 2/neu is a member of epidermal growth factor receptor family . The family consists of four receptors, they are HER-1, HER-2, HER-3 and HER-4. These receptor have tyrosine kinase activity and regulate cell growth, survival . They are responsible for cellular proliferation and differentiation and also plays a important role in the pathogenesis of numerous cancers.

Her 2/neu receptor is a transmembrane glycoprotein, gene located at the long arm of chromosome 17. Once ligand binds to the extracellular domain of this receptor, it undergo dimerization and phosphorylates their intracellular domanis. Her 2/neu can be activated in the absence of ligand by heterodimerization with other family members. Homo or heterodimerization leads to autophosphorylation of tyrosine residues and initiates of signaling pathways. These pathways included mitogen – activated protein kinase (MAPK), phosphatidylinositol -4-5, - bisphosphate 3 kinase (PI3K) and protein kinase C ( PKC) resulting in cellular proliferation , differentiation (Figure 4) . Her2 neudimerization causes rapid degradation of p27 . p27 is a cell cycle inhibitor. This will lead to uncontrolled cell cycle progression ⁽⁵⁹⁾. (Figure 4).

(54)

Figure 4

H E R 2

E G F R

H E R 2

H E R 3

H E R 2

H E R 4

Ras

Raf

MAPKK

MAPK PI3K

AKT mTOR

PLC

PKC

Nuclear transcription

Proliferation Survival Cell cycle progression

CYTOPLASM

NUCLEUS EXTRACELLUAR SPACE

(55)

HER 2/neu overexpression in cancers:

Her 2/neu over expressed in many cancers. It is over expressed in carcinoma of breast, stomach, ovary, endometrium, colon, bladder, lung, uterine cervix, head

& neck and esophagus ⁽⁶⁰⁾. Apart from its role in the development of cancers it has been identified for targeted therapy.

Her- 2/neu overexpression has been reported in 4% to 69%⁽⁶¹⁾. Multiple studies showed overexpression of HER 2 have been associated more frequently with non endometrioid carcinomas ⁽⁴⁷⁾.

Her-2/neu expression is associated with advanced stage, grade III and more than 50% of myometrial invasion. It is a negative prognostic factor in endometrial carcinoma⁽⁴⁷⁾. Her 2/ neu overexpressed tumors respond to the targeted drug (Trastuzumab)⁽⁶²⁾.

(56)

MATERIALS AND METHODS

Endometrial carcinoma reported between the period from January 2010 to December 2014 in the department of pathology of this institute were considered for the study. The study included the cases of endometrial carcinoma reported on both curetting and hysterectomy specimens. On samples where both hysterectomy and curettings were available, hysterectomy samples alone were taken for the study though the initial diagnosis was on curettings for staging reasons.

The clinical details like patients age, parity, menstrual status, clinical staging and gross specimen details were taken from the pathology requisition slips and from the medical records department of this institute after obtaining permission from the concerned authorities and institute human ethics committee clearance.

The Hematoxylin and Eosin slides of these cases were analyzed for assessing histopathological type of the tumor and grading of the tumor. As a rule of thumb the slide should have minimum of 60-70% well preserved tumor area with less than 10% of necrotic area for immunohistochemistry. This was done to prevent staining artifact while doing immunohistochemical studies. Four thin (4 to 5 micron) sections were taken from representative blocks to perform immunohistochemical study on ER, PR , Bcl-2 and Her 2/ neu.

(57)

FIGURE 5: THE STUDY PLAN

ALL CASES DIAGNOSED AS ENDOMETRIAL CARCINOMA JANUARY 2010 – DECEMBER 2014

BOTH CURETTING AND HYSTERCTOMY BLOCKS INCLUDED

CURETTING BLOCKS OF PATIENT WITH HYSTERECTOMY BLOCKS WERE

EXCLUDED

SLIDES & BLOCKS WERE RETRIEVED FROM ARCHIVES OF HISTOPATHOLOGY

H & E SLIDES EXAMINED FOR HISTOLOGIC TYPING AND

GRADIG OF TUMORS

ER, PR , BCL-2 & HER 2 NEU WERE PERFORMED

ALL MARKERS EXPRESSION WERE ASSESED

BCL2 EXPRESSION WAS CORRELATED WITH ER, PR & HER2 NEU EXPRESSION

(58)

Immunohistochemistry was done using supersensitive HRP (Horse Radish Peroxidase enzyme) detecting system. The following table shows the clones of various primary antibodies used during the study.

TABLE 4

THE MARKERS, CLONE OF THE PRIMARY AND CONTROLS USED

MARKERS CLONE COMPANY CONTROL

ER Monoclonal ER alpha

EP1

PATHNSITU CARCINOMA

BREAST

PR Monoclonal PR – EP2 PATHNSITU CARCINOMA

BREAST

HER 2 NEU Monoclonal Her2/ erb 2- EP 3

PATHNSITU CARCINOMA

BREAST

BCL-2 BCL-2 EP 36 PATHNSITU LYMPHNODE

(59)

The immunohistochemistry procedure is similar for all markers and it as follows.

PRINCIPLE :

In an immunohistochemical reaction, the specific antigen present in the tissue is detected in a two stage process

1. In the first step , the primary antibody is bound to its targeted epitope in the tissue

2. Followed by the detection of bound primary antibody by using secondary antibody by a colorimetric reaction.

The secondary antibody is bound to a dextran polymer with the help of horseradish peroxidase enzyme. The reaction involving binding of secondary antibody with the primary antibody is amplified by the use of a suitable chromogen 3,3’diaminobenzidine tetra hydrochloride (DAB) and which is responsible for the colour.

(60)

 STEP 1:

RETRIEVAL OF THE ANTIGEN⁽⁶³⁾:

Routine processing of tissue with formalin, makes antigen site gets obscured.

The antigen sites are masked by cross linking actions of formalin. The antigen has to be exposed for binding with antibody. So the process by which the antigenic site is exposed is called as antigen retrieval.

The various methods used for this are:

1. Pressure cooker method

2. Microwave method

3. Proteolytic digestion method

We use pressure cooker method for our study. In this method the antigenic site are unmasked by the action of both pressure and heat. Before this process, the slides were dewaxed and hydrated through graded alcohols.Antigen retrieval by pressure cooker is done for 10 minutes in EDTA buffer at an alkaline pH of 9.

Reagents used are:

 Ethylene Diamine Tetra-acetic Acid ( EDTA) buffer at pH 9.

 3% Hydrogen peroxide in distilled water.

Hydrogen peroxide blocks the endogenous peroxidase action, therefore it prevents nonspecific background staining.

(61)

 A solution of 0.01 M Phosphate Buffered Saline (PBS) is prepared with a pH value of 7.6. The solution is the combination of following substances in a litre of distilled water.

1. Dibasic sodium phosphate , anhydrate 17.5 g

2. Monobasic potassium phosphate, anhydrous 2.5 g

3. Sodium chloride 17.0 g

 Blocking reagent:

Casein in buffered saline with 15 milli mole sodium azide is used for blocking nonspecific protein binding

 STEP 2

 Primary antibodies against Bcl -2, Her 2/ neu, ER, PR

 STEP 3

 Poly HRP reagent - Horse Radish Peroxidase enzyme

 STEP 4

 DAB (3,3’DiaminoBenzidine tetra hydrochloride) is used as a chromogen and causes permanent coarse brown precipitate.

(62)

 STEP 5

 Harris Haematoxylin as counter stain

 DPX (Distyrene Phthalate Xylene )-mountant.

PROCEDURE:

With the help of microtome, 4-5 micron thin sections were floated on the slides which were coated with Poly L Lysine in a water bath maintained at 37 degree Celsius⁽⁶⁴⁾. The immunohistochemical staining procedure is carried as followed:

 Deparaffinization of slides by xylene were done.

 Deparaffinised slides were hydrated through graded alcohols.

 Washed in running tap water for one minute.

 Antigen retrieval is done by using pressure cooker method in EDTA buffer with pH of 9 for 10 minutes.

 Fast cooling under tap water.

 Washed in PBS buffer at pH 7.6 for 5 minutes.

 The slides are immersed in 0.3% hydrogen peroxide for 20 minutes. It inhibits the background staining due to endogenous peroxidase enzyme.

 The slides are washed with PBS buffer for 5 minutes.

(63)

 Slides are incubated in blocking solution for 10 minutes. Blocking solution prevents the nonspecific reaction of the reagent antibody with other tissue antigen.

 Slides are washed with PBS buffer three times for 5 minutes.

 Slides are incubated with BCL-2 primary antibody for one hour.

 To enhance the signal intensity, the sections were treated with superenhancer for half an hour.

 Wash with PBS buffer three times for 5 minutes.

 The sections were covered with super sensitive polymer HRP for 30 minutes.

 Wash the slides with PBS for three times.

 Chromogen DAB is added and incubated for 8 minutes

 Slides are washed with PBS buffer thrice for 5 minutes.

 Harris Hematoxylin stain is used as counter stain for one minute.

 Washed in running tap water

 Sections are cleared with xylene and mounted with DPX

(64)

• TISSUE PREPARATION & ANTIGEN RETRIVAL STEP 1

• PRIMARY ANTIBODY STEP 2

• SECONDARY ANTIBODY STEP 3

• DAB - CHROMOGEN APPLICATION STEP 4

• COUNTER STAIN & MOUNT STEP 5

(65)

The stained slides were examined for the expression of Bcl-2, Her 2 neu, ER, and PR and compared with controls. The intensity of the staining was scored based on many studies.

Evaluation of ER and PR status was performed according to the method described by Carcangiu et al ⁽⁵⁴⁾.

Table 5 : ER, PR SCORING

ER and PR Score Stained cells Category ( score=P+I)

Proportion (P)

1

2

3

- 25%

26% - 75%

>75%

Category 1: Scores 1 and 2

Intensity ( I ) 1

2 3

Absent to weak staining

Strong staining Very strong staining

The combined score of more than 2 (Categories 2 and 3 ) was considered immunopositive

(66)

Based on study conducted by G. Guset et al ⁽⁶⁾ an arbitrary scoring system was drawn up for quantifying the expression of Bcl-2.

Table 6: BCL-2 SCORING

SCORE DESCRIPTION

0 Negative: no or fewer than 5% positive cells

1+ Positive immunoreaction in 5-20% of tumor cells

2+ Positive immunoreaction in 30-50% of tumor cells

3+ Positive immunoreaction in more than 50% of tumor cells

Immunohistochemical Expression of Bcl-2 in Endometrial Carcinoma and Its Correlation with Hormone Receptor Status and Transforming Growth Factor

M.D in PATHOLOGY

TABLE OF CONTENTS

Figure 2

SURFACE EPITHELIUM

GENOMIC DAMAGE

ENDOMETRIAL GLANDULAR

DYSPLASIA

INVOLUTION SEROUS EIC

INVASION

UPSC