MAY 2020

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AN IMMUNOHISTOCHEMICAL STUDY OF ANDROGEN RECEPTOR AND BETA-CATENIN IN TRIPLE NEGATIVE

BREAST CANCERS

Dissertation submitted in

partial fulfilment of the requirements for the degree of

M.D. PATHOLOGY

BRANCH – III

INSTITUTE OF PATHOLOGY MADRAS MEDICAL COLLEGE

CHENNAI – 600 003

m y

THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY CHENNAI

MAY 2020

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CERTIFICATE

This is to certify that this Dissertation entitled “AN IMMUNOHISTOCHEMICAL STUDY OF ANDROGEN RECEPTOR AND BETA-CATENIN IN TRIPLE NEGATIVE BREAST CANCERS” is the bonafide original work of DR. VANI K in partial fulfilment of the requirement for M.D., (Branch III) in Pathology examination of the Tamilnadu Dr.M.G.R Medical University to be held in May 2020.

Prof. Dr. Sudha Venkatesh, M.D., Professor of Pathology

Institute of Pathology,

Madras Medical College/RGGGH, Chennai- 600003

Prof. Dr. Bharathi Vidhya JayanthiM.D., Director and Professor of Pathology,

Institute of Pathology, Madras Medical College, Chennai -600003

Dr. R. Jayanthi,M.D.FRCP (Glasg) DEAN

Madras Medical College &

Rajiv Gandhi Govt. General Hospital, Chennai – 600003.

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DECLARATION

I, DR. VANI K, solemnly declare that the dissertation entitled

“AN IMMUNOHISTOCHEMICAL STUDY OF ANDROGEN RECEPTOR AND BETA-CATENIN IN TRIPLE NEGATIVE BREAST CANCERS” is the bonafide work done by me at the Institute of Pathology, Madras Medical College under the expert guidance and supervision of Prof. Dr. SUDHA VENKATESH, M.D., Professor of Pathology, Institute of Pathology and DR.

RAMYA, M.D., Assistant professor of Pathology, Institute of Pathology, Madras Medical College. The dissertation is submitted to the Tamilnadu Dr.M.G.R Medical University towards partial fulfilment of requirement for the award of M.D., Degree (Branch III) in Pathology.

Place: Chennai

Date: DR. VANI K

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ACKNOWLEDGMENT

I express my sincere thanks to Prof. Dr. JAYANTHI R, M.D.,FRCP (Glasg), Dean, Madras Medical College and Rajiv Gandhi Government General Hospital, for permitting me to utilize the facilities of the Institution.

I take the opportunity to express my gratitude to Prof. Dr. BHARATHI VIDHYA JAYANTHI, M.D., Director and Professor, Institute of Pathology, Madras Medical College, Chennai for her keen interest, constant encouragement and valuable suggestions throughout the study.

I am extremely thankful to Prof. Dr. SUDHA VENKATESH, M.D., Professor of Pathology, Institute of Pathology, Madras Medical College, for her constant encouragement, wholehearted support, valuable suggestions and expert guidance throughout the study, without which this study would not have ever been possible.

I am truly thankful to Dr. SURESH M.D., D.M., Assistant Professor, Medical Oncology, for his valuable suggestions.

I am truly thankful to Prof. Dr.Padmavathi M.D., , Prof.Dr. Geetha Devadas M.D DCP., Prof.Dr.M.P.Kanchana M.D., Prof. Dr.S.Pappathi M.D., Prof. Dr.Rajavelu Indira M.D., Prof.Dr.Selvambigai M.D., Prof.Dr.K. Rama, M.D, Prof. Dr. Sheeba, M.D, for their valuable suggestions and encouragement throughout the study.

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I express my heartfelt sincere thanks to all my Assistant Professors for their help and suggestions during the study.

I am thankful to my colleagues, friends, technicians and staff of the Institute of Pathology, Madras Medical College, Chennai for all their help and support they extended for the successful completion of this dissertation. My sincere thanks also go to all the patients and their families who were co-operative during the course of this study.

Last but not the least, I am grateful to my family members and friends for their constant support and belief in me.

Above all I thank the ALMIGHTY, for everything that he has given to me.

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CERIFICATE - II

This is to certify that this dissertation work titled “AN IMMUNOHISTOCHEMICAL STUDY OF ANDROGEN RECEPTOR AND BETA-CATENIN IN TRIPLE NEGATIVE BREAST CANCERS” of the candidate Dr. VANI K with registration Number 201713009 for the award of M.D PATHOLOGY (Branch-III). I personally verified the urkund.com website for the purpose of Plagiarism Check. I found that the uploaded thesis file contains from introduction to conclusion and result shows 3 percentage of plagiarism in the dissertation.

Guide & Supervisor sign with seal

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ABBREVIATION

HPE : Histopathological examination IHC : Immunohistochemistry

AR : Androgen receptor

HER2 : Human epiudermal growth factor receptor

IBC NOS : Invasive Breast carcinoma not otherwise specified TNBC : Triple negative breast cancer

DCIS : Ductal carcinoma in situ FCD : Fibrocystic disease

IDP : Intraductal pappillomatosis

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INTRODUCTION

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INTRODUCTION

Breast cancer is the most common cancer worldwide in women. It remains a serious global health issue. It is the most common cause of cancer deaths in developing countries. Recent decline in mortality in breast cancer in developed countries is attributed to increased public awareness, advances in breast imaging, screening and new innovations in breast cancer therapy. Mortality rates will be reduced if diagnosed and treated early. Breast cancers exhibit varying behavior with regard to recurrence metastasis and response to treatment.

Triple negative breast cancer is clinically an aggressive subgroup of breast cancer which accounts for 10 to 20 % of breast tumours.¹ They pose a great diagnostic challenge as they are very aggressive and have high metastatic potency.

Triple negative breast cancers do not respond to endocrine therapy or routinely available targeted therapies. Commonly used prognostic markers for breast cancer are estrogen and progesterone receptors. Unfortunately in triple negative breast cancer, the established endocrine therapies are ineffective and highlights an urgent need for additional therapeutic targets.

Androgen receptor is a nuclear steroid hormone receptor family member.

Androgen receptor is expressed by apocrine cells and luminal epithelial cells of breast.² Tumours arising from these cells express AR and can be targeted through AR targeted therapy.

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Therapeutic options for triple negative breast cancers are limited because no pathway specific targets and associated biomarkers have been established.

Ligand driven wnt-beta-catenin pathway is activated in triple negative breast cancers and associated with poor prognosis.

In this study of 40 cases which included Estrogen Receptor, Progesterone Receptor HER 2 neu (Human Epidermal growth factor Receptor), negative breast cancers. We have attempted to evaluate the expression of Androgen Receptor and Beta catenin by IHC.

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AIMS AND OBJECTIVES

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AIMS AND OBJECTIVES

1. To evaluate the status of androgen receptor and beta-catenin in triple negative breast cancers.

2. To correlate the immunohistochemical expression of these markers with histological grade

3. To correlate the relation between androgen receptor and beta catenin

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REVIEW OF LITERATURE

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REVIEW OF LITERATURE

Breast carcinoma is the most commonly detected malignancy in women. It is the most common female cancer worldwide representing nearly a quarter (25%) of all cancers with an estimated 1.67 million new cancer cases³.The incidence of breast cancer has significantly increased and surpassed cervical cancer although cervical cancer still remains most common in rural india.³

Breast carcinoma is the most common malignancy in women with 6.6% of cases detected below the age of 40.⁴ Invasive carcinomas are morphologically subdivided according to their growth patterns and degree of differentiation.

Histological tumor grade is based on the degree of differentiation of the tumor tissue⁵

Nottingham grading system is based on the evaluation of three morphological features: (a) degree of tubule or gland formation, (b) nuclear pleomorphism, and (c) mitotic count

Fig 1: Grading of breast cancer

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Histological grade of breast cancer assessed by the Nottinghamnt Grading System. (a) Grade I well-differentiated tumor that demonstrated tubule formation (>75%), mild degree of nuclear pleomorphism, and low mitotic count. (b) A moderately differentiated tumor (grade 2). (c) A poorly differentiated (grade 3) tumor with a marked degree of cellular pleomorphism and frequent mitoses and no tubule formation (<10%).

EPIDEMIOLOGY:

The most frequent cancer globally in women is breast cancer which represents the second leading cause of cancer death among women . India is going through epidemiologic transition with breast carcinoma showing a rapid rise especially in urban centres. Breast cancer incidence is increasing rapidly as a result of changes in dietary habits and increasing life expectancy, acting in concert with genetic factors.⁶

In India, the crude incidence rate of breast carcinoma is 85/100,000 women / year.⁷The death per incident ratio is highest in India, with 50%, compared to 30% in China and 18% in the US.

Histology and molecular analysis showed breast carcinoma is a heterogeneous disease composed of morphologically and genetically distinct entities with different molecular profile, behaviour and response.

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6 RISK FACTORS:

1.AGE:

Age is one of the most important risk factor for breast cancer, because the incidence of breast cancer is highly related to the increasing age.⁸

2.GENETICS:

Breast cancer associated gene 1 and 2 (BRCA1 and BRCA2) are two anti- oncogenes for breast cancer risk. BRCA1 and BRCA2 are located on chromosome 17q21 and 13q12, respectively. Totally, about 20-25% of hereditary breast cancers and 5-10% of all breast cancers are caused by BRCA1/2 mutations ^.9,10 A meta-analysis by Chen showed that the breast cancer risk ratio in women older than 70 years carrying BRCA1 or BRCA2 mutations was 57% and 49%, respectively.¹¹

Apart from breast cancer, this mutation also have increased risk of ovarian cancer and pancreatic cancer. Those with inherited mutations in other two genes, p53 and PTEN, are associated with familial syndromes (Li-Fraumeni and Cowden's respectively) that include a high risk of breast cancer .¹²

3.FAMILY HISTORY:

Females with first degree relative with breast cancer have a risk of 2-3 times that of the general population, and if the relative was affected at an early age the risk is further increased.

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7 4. REPRODUCTIVE HISTORY:

Increased risk is seen with early menarche,nulliparity, late age at first birth, and late menopause. Breast cancer is rare in those who have undergone oophorectomy before the age of 35 years and reduces the risk to one-third.

Women who have their first child before the age of 18 years have only 1/3rd the risk of those whose first child is delayed until age 30

5. ENVIRONMENTAL FACTORS:

a) Diet: High dietary fat is one of the significant predisposing factors for breast cancer.

b) Alcohol increases the risk of breast cancer.

c) Radiation: Women treated with mantle radiation for Hodgkins lymphoma have 20% to 30% risk of breast carcinoma development

6. OTHER FACTORS:

a) Carcinoma of the contralateral breast (or) endometrium is an increased risk of breast cancer.

CARCINOGENESIS:

Breast cancer develops by a multistep process. This process involves various steps which includes transformation of normal cells via the steps of hyperplasia, premalignant change and in situ carcinoma.¹³

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Molecular interdependencies elucidates the development of primary breast cancer. Also it leads to its progression, and its formation of metastases which is the main focus for new strategies targeted at prevention and treatment.

Amplifications, deletions and gene mutations are the somatic changes in the genome of breast cancer cells. ⁴

Fig.2: Proposed precursor-carcinoma sequences in breast cancer

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9 CLINICAL PRESENTATION

50% of breast carcinoma are located in the superior and outer quadrant, 15% in the superior and inner quadrant, 10% in the inferior and outer quadrant, 5% in the inferior and inner quadrant, 17% in the central quadrant and 3% are diffuse.

RADIOLOGICAL IMAGING:

1. Mammogram:

The widespread use of mammography brought about a drastic change in the diagnosis of breast cancer. ^15,16 Mammographic screening was first implemented in the 1980s for detecting small non palpable carcinoma that were asymptomatic. With increasing age, the radio dense and fibrous breast tissue of youth was replaced by the radiolucent fatty tissue that increases the mammographic specificity and sensitivity. The probability of mammographically detected cancerous lesion rises from 10% at 40 years to 25% at 35 years of age.

The primary signs of mammographically detected carcinomas include density and calcification.

A. Mammographic Density

Mammographic density is produced frequently by invasive carcinoma, fibroadenoma or cyst. Most tumors are denser radiologically compared to the

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adjacent normal breast parenchyma. Mammography is valuable for detecting tiny, clinically non palpable cancer.

Calcification:

Calcification is seen in the areas of necrosis, hyalinised stroma or secretion.

The incidence of calcification in breast carcinoma is 50-60% ^17,18Whereas the incidence of calcification in benign breast disease is 20%.

Hyalinised fibroadenomas, apocrine cysts and sclerosing adenosis are associated with benign calcification.

Calcification in malignancy are usually tiny, numerous, irregular and clustered.

DCIS is most frequently detected as a calcification in mammogram.

They are deposited in linear branching pattern.

Small sized Invasive Ductal adenocarcinomas rarely present with calcification unaccompanied by mammographic radiodensity. Lymph node metastasis is rare in these cases.

2. USG:

USG can distinguish between cystic & solid lesions.

It can delineate the borders more precisely in case of solid masses.

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11 3. MRI:

It detects breast carcinomas by uptake of contrast agents owing to increased vascularity of the tumor.

It is helpful for screening high risk women and those with dense breast.

To determine the extent of involvement of chest wall by cancers that are locally advanced. For evaluating cases with rupture of breast implants.

TISSUE SAMPLING METHODS

 Trucut Biopsy

 Incision Biopsy

 Excision Biopsy

 Radical and Modified Radical Mastectomy

 FNAC

CLASSIFICATION OF BREAST CANCERS

Breast carcinoma is classified clinically based on tumor size, lymph node status, local extent and distant spread, morphologically based on histological type and grade, At Molecular level, according to hormone receptors and HER2neu status.

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MOLECULAR CLASSIFICATION OF BREAST CANCER: 19,20,21,22

Luminal A:

This phenotype is seen in 40% - 50% of the IDC NOS type cancer. It includes ER positive and HER2neu negative tumor. Most of the tumors are moderately to well differentiated with increased expression among postmenopausal female.

These tumors are slow growing and respond well to hormonal therapy. But response to standard chemotherapy is seen in only minimal number of cases.

Luminal B:

This phenotype is seen in 15% - 20% of IDC-NOS type cancer. They are triple positive tumors with expression of ER, PR & HER2neu.

They are of higher grade with increased proliferative potential.

Increased frequency of nodal metastasis is seen.

Responds well to chemotherapy.

Normal Breast Like:

This phenotype accounts for 6% - 10% of IDC NOS type cancer.

This is a minor group consisting of well differentiated ER positive &

HER2neu negative cancers. They show similar gene expression pattern

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13 Basal Like:

This phenotype accounts for 13% - 25% of IDC NOS type cancer. These tumors are characterized by the absence of PR, ER & HER2neu expression with expression of basal myoepithelial markers like P63, PCadherin markers and of progenitor cells / putative stem cells (CK 5/6)

This group is referred as “TRIPLE NEGATIVE” Carcinoma. ^23,24 Tumors included in this category are medullary & metaplastic carcinomas. Breast carcinomas harbouring BRCA1 mutation usually belong to this group.

They are of higher grade with increased proliferative potential and aggressive clinical course.

They are frequently associated with visceral and CNS metastasis.

Complete response following chemotherapy is seen in only 15-20% of cases.

HER2neu Positive:

This phenotype is seen in 7% - 12% of IDC NOS cancers.

It includes carcinomas with HER2neu over expression and ER / PR negativity.

In 90% of cases, over expression is mainly due to amplification of the DNA segment on chromosome 17q21 that harbors the HER2neu gene and varying number of adjacent genes.

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HER2neu assay that include measurement of gene copies number by FISH, mRNA level, by gene assay and protein by IHC are all deranged in most of these cancers.

They are generally poorly differentiated with increased proliferative potential and associated with increased frequency of CNS metastasis.

PROGNOSTIC FACTORS:

Prognostic factors are important for predicting the likely outcome of the cancer, and appropriate treatment

1) AGE OF THE PATIENT

Prognosis is better if age of the patient is less than 50 years of age.

Prognosis declines after the age of 50 years

2) SIZE

Size is an important prognostic factor and studies shows good correlation between nodal status and survival rate. ^25,26 Size is one of the two criteria for the definition of minimal breast carcinoma, which includes all insitu carcinomas regardless of size and invasive carcinomas of <1cm in diameter.

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15 SITE:

Tumors located in the medial quadrant were associated with higher risk of (50%) relapse than laterally located tumor. ²⁷

CYTOARCHITECTURAL TYPE

There is no significant difference in prognosis between invasive ductal and lobular carcinoma .²⁸Morphological variants like Tubular, Mucinous, Medullary, Papillary, Cribriform, Adenoid cystic carcinoma and Secretary Carcinoma have good prognosis. ²⁹

Variants like Inflammatory, Metaplastic, Squamous cell carcinoma, Neuroendocrine and Signet ring cell carcinoma are aggressive tumors with bad prognosis. ³⁰

PRESENCE OR ABSENCE OF INVASIVENESS

The amount of insitu component correlates with the incidence of multicentricity and with the probability of occult invasion. ³¹

In situ ductal malignancies of the comedocarcinoma type can be associated with metastases .

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16 TYPE OF MARGINS:

Tumors with infiltrating margins have a worse prognosis than tumors with pushing margins. ^32,33

TUMOUR GRADE:

The widely accepted powerful indicator of prognosis in breast cancer is histologic grading. Currently employed tumor grading systems for breast cancer combine nuclear grade, tubule formation and mitotic rate. The original Scarff- Bloom-Richardson (SBR) system is the most common grading system used in United States and the Black method which emphasizes nuclear grading and excludes consideration of tubules as a criteria .³⁴Europe uses the Elston-Ellis modification of the SBR grading system (Nottingham grading system) and is becoming increasingly popular in the US .³⁵

SKIN INVASION:

Breast carcinomas with skin infiltration are associated with decreased survival rate. ³⁶

NIPPLE INVASION:

Nipple involvement by carcinomas is associated with increased incidence of axillary metastasis. ³⁷

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17 TUMOR NECROSIS:

Tumor necrosis is associated with an increased nodal metastases &

reduced survival rates .³⁸ This feature is usually associated with tumors of high histologic grade. ³⁹

VASCULAR INVASION:

Vascular invasion is associated with poor prognosis.⁴⁰

LYMPHOVASCULAR INVASION:

Presence of lymphovascular invasion within the breast is associated with more risk of tumour recurrence.^41,42

LYMPH NODE STATUS:

Axillary lymph node involvement is an important prognostic factor.

umber of axillary nodes involved, level of the node and amount of tumor cells in the node, presence or absence of tumor cells in the efferent blood vessels have important implication in patient’s survival. ⁴³

METASTASIS:

Presence of distant metastasis have poor prognosis. The timing and location of metastasis is also influenced by the tumor type. ^44,45

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18 BRCA-1 STATUS:

The Breast carcinomas with BRCA 1 mutation carriers are associated with overall poor survival ⁴⁶

HORMONE RECEPTORS:

The presence of estrogen and progesterone receptors in an invasive breast carcinoma is both prognostic and predictive. The c-erbB-2 (HER2/neu) proto- oncogene is located on 17q21 and encodes an Mr 185,000 transmembrane glycoprotein, p185HER2, with intrinsic tyrosine kinase activity homologous to the epidermal growth factor receptor . It is overexpressed in approximately 30%

of human breast tumors .⁴⁷ Overexpression is associated with increased tumor aggressiveness, increased rates of recurrence, and increased mortality in node- positive patients, while the influence in node-negative patients is more variable

TRIPLE NEGATIVE BREAST CANCERS:

Triple negative breast cancer (TNBC)means that the breast cancer cells have tested negative for human epidermal growth factor receptor 2 (HER-2), estrogen receptors (ER), and progesterone receptors .As the tumor cells lack the hormone receptors common treatments like hormone therapy and drugs that target estrogen, progesterone, and HER-2 are ineffective. TNBC is characterized by its unique molecular profile, aggressive nature, distinct metastatic patterns and lack of targeted therapies. It is estimated that out of the worldwide breast cancer

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burden, approximately 170,000 cases are TNBC and account for ~10-20% of invasive breast cancers.^48,49

This warrants further studies on the intensification of chemotherapy and identification and development of targeted therapy aimed at decreasing recurrences and improving survival in this patient population.⁵³Triple negative breast cancer is still treated by chemotherapy which is an effective option. Triple negative breast cancer responds better to chemotherapy in the earlier stages .

It has been observed that a high percentage of BRCA1- associated hereditary and sporadic breast cancers are triple negative and express a high proportion of basal like cytokeratins (CK5,14,17), as well as P-Cadherin and HER1/EGFR ^50,51 TNBC with BRCA1 gene mutations may be more sensitive to agents that cause DNA damage, such as Cisplatin ⁵²

The key to developing targeted therapies for TNBC will depend on understanding the molecular mechanism for the development of these cancers.

TNBCs have a rapid clinical course and early recurrences inspite of timely medical intervention due to the aggressive tumor expression profiling.

Lehmann et al. identified seven molecular subgroups: unstable; basal-like 1;

basal-like 2; immunomodulatory; mesenchymal (MES)-like; mesenchymal stem- like (MSL); and luminal androgen receptor (LAR)⁵⁴. The Immunomodulatory and MSL subtypes have recently been refined.⁵⁵ Burstein et al. applied non-negative

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matrix factorization and defined four subgroups: basal-like immune active; basal- like immune suppressed; mesenchymal; and luminal AR.⁵⁶^,57

Accelerated growth, high recurrence rates, and frequent metastasis characterize the aggressiveness of TNBC and result in poor long-term patient survival.⁵⁸

ANDROGEN RECEPTOR:

Androgen receptor (AR) is expressed by IHC widely in all subtypes of breast cancer. AR expression is more commonly found in well-differentiated, relatively indolent cancers . Signalling pathways of AR has a role in endocrine resistance, and clinical trials have been completed with antiestrogens and antiandrogens combinations such as abiraterone acetate and enzalutamide.

Trials are also ongoing with the combination of antiandrogens and anti-HER2 agents in HER2+ metastatic breast cancer. Anti androgen therapy remains the most promising field in triple negative breast cancers with the identification of a molecular subgroup characterized by high expression of AR at the mRNA level.

13%–37% of triple negative breast cancers express androgen receptor .^59,60 The biological and therapeutic role of AR independent of other hormone receptors can be studied in this subtype as ER and PR are absent in TNBC. AR positivity in TNBC is associated with older age at presentation, coinciding with the high circulating levels of androgens seen in postmenopausal breast cancer patients.⁶¹ Luminal Androgen Receptor Subtype(LAR).

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The Luminal AR is a subgroup expressing androgen receptors. By demonstrating AR positivity in breast cancer it is a prognostic and predictive factor. ^62-64The luminal androgen receptor subtype contains pathways that regulate steroid synthesis, porphyrin metabolism and androgen/estrogen metabolism.⁶⁵ In this subtype, the androgen receptor is heavily expressed, with an expression 10- fold greater than the other subtypes.⁶⁶The LAR subtype represents approximately 11% of TNBCs ⁶⁶.Positive AR expression was seen in 75% of luminal A, 70%

of B, 47% of Her2 like and 31% of TNBC^67.

Androgen receptor (AR) which is a steroid hormone receptor has emerged as a possible biomarker for cancer treatment in ER positive patients that fail endocrine therapy and in TNBC patients ⁶⁸

The LAR subtype is associated with the expression of genes involved in the metabolism of androgens and estrogens. The LAR subtype is associated with dismal response rates to cytotoxic chemotherapy .⁶⁹ Lehmann et al. also demonstrated that TNBCs belonging to the LAR subtype had significantly higher levels of AR expression assessed by IHC leading to its use as a surrogate marker for the LAR subtype ⁷⁰ The AR positive subtype has better prognosis and less chemotherapy responsiveness.

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AR is involved in cell cycle regulation and Epithelial-to-Mesenchymal Transition. Clinical data demonstrate the efficacy of antiandrogen therapies for AR positiveTNBC

Fig 3: Androgen Receptor in TNBC

Beta-catenin is key regulatory protein involved in cell adhesion and signal transduction through the wnt pathway.Wnt signal transduction pathway is activated in several tumor types. Thus targeting beta catenin may have important implications in triple negative breast cancers therapy.

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Fig 4: WNT signalling

The canonical Wnt signal transduction pathway is activated in several tumor types, including colorectal and breast cancer .^71-73 The major effector of the canonical Wnt pathway, β-catenin, is stabilized in tumors primarily via Wnt ligand overexpression, down-regulation of Wnt ligand antagonists, or loss of the APC tumor suppressor .⁷⁴As a consequence of its stabilization, β-catenin translocates to the nucleus where it controls gene expression through its association with members of the T cell factor (TCF) family of transcription factors. Some of the β-catenin/TCF transcriptional targets implicated in tumor initiation and progression include the cell cycle regulators cyclin D1 ⁷⁵and c- Myc ⁷⁶, stem cell gene Bmi-1 ⁷⁷, matrix metalloproteinase Mmp-7 ⁷⁸, and Wnt pathway component Axin2 ⁷⁹. Wnt pathway is more frequently activated in

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TNBC than in other breast cancer subtypes . ^{80-82 .}Moreover, nuclear and cytosolic accumulation of β-catenin is associated with reduced overall survival in all breast cancer patients .

Reis-Filho’s team reported that Wnt-beta-catenin pathway activation is (1) preferentially seen in triple negative breast carcinomas, (2) associated with poor clinical outcome and (3) unlikely to be driven by CTNNB1 mutations in breast cancers. ⁸²

Metastatic progression is the single most important cause of poor outcomes in TNBC patients. Tumor cells in the course of their evolution acquire genetic alterations that triggers the metastatic potency via deregulation of different signaling pathways . The upregulation of Wnt signaling in TNBC patients is associated with metastasis .⁸³ TNBC is functionally associated with metastasis- associated phenotypes.

Wnt signaling pathway and its components are activated in various cancers where they regulate the cancer stem cells that lead to development and progression of tumor. The role of the pathway components is definitive in colorectal cancer and has now emerged for the breast cancer also. Wnt signaling could be targeted in TNBC which so far does not have any targeted therapy. ⁸⁴

β-catenin membranous expression was observed in all subtypes of breast cancer and its expression decreases with tumor progression. Cytosolic and nuclear localization of β-catenin was associated with other markers of the basal-

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like phenotype, including nuclear hormone receptor and HER2 negativity, cytokeratin 5/6 and vimentin expression, and stem cell enrichment. Subcellular localization of β-catenin was associated with a po or outcome.⁸⁵ It is possible that through further characterization of the role of Wnt/β-catenin signaling in basal-like tumors, β-catenin will emerge as a valuable therapeutic target for triple negative breast cancer subtype.

SCORING OF ANDROGEN RECEPTOR:

AR expression was semi-quantitatively analyzed according to the percentage of cells showing nuclear positivity: 0, 0%; 1+, 1%–29%; 2+, 30%–

69%; 3+, ≥70%. Scores ≥1 were considered positive, and a score of 0 was negative ^86,87,88 AR expression also had a significant effect on cancer stem cells.

Patients with AR-positive TNBCs survived longer after recurrence than those with AR-negative TNBCs. There is a difference in malignant potential between AR- positive and -negative TNBC. TNBCs that are positive for AR expression have a significantly lower rate of pathological complete response (pCR) in neoadjuvant chemotherapy (NAC) and are chemotherapy-resistant .⁸⁹AR expression may be useful as a subclassification marker for good prognosis in TNBC, and that AR- positive TNBCs may be responsive to anti-androgen endocrine therapy. AR is a potential target for breast cancer .⁹⁰AR expression correlates with better prognosis for both primary and metastatic cancer.

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β-catenin expression was scored semiquantitatively according to the following criteria:⁹¹

 A score of 0 if <1% of morphologically unequivocal neoplastic cells discretely expressed cytoplasmic β-catenin

 A score of 1+ if ≥1 and <10% of morphologically unequivocal neoplastic cells discretely expressed cytoplasmic β-catenin

 and a score of 2+ if ≥10% of morphologically unequivocal neoplastic cells discretely expressed cytoplasmic β-catenin

 Grades 1+ and 2+ were considered β-catenin positive.

IMMUNOHISTOCHEMISTRY

Immunohistochemistry was first described by Dr. Albert Coons in 1941.

Since then numerous advancements in the technique have been made.⁹² The most commonly used technique is the Peroxidase-antiperoxidase immune complex method developed by Sternberger in 1970. The newer biotin-avidin immunoenzymatic technique was developed by Heitzman and Richards in 1974.^93,94

USES OF IHC IN BREAST PATHOLOGY

1. The use of Myoepithelial markers to assess stromal invasion.

2. To differentiate between different types of breast cancer. Eg. E-cadherin helps to differentiate between ductal and lobular carcinoma.

3. To differentiate between precursor lesions and malignancy.

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Eg. HMWCK helps to distinguish between Usual Ductal Hyperplasia and Ductal carcinoma insitu.

4. To find the site of origin in metastatic cancers.

5. To detect sentinel lymphnode metastasis.

6. Assessment of Estrogen and Progesterone receptor status and HER2neu overexpression using specific antibodies to receptor proteins.

7. Evaluation of Metaplastic carcinoma from mesenchymal lesions.

ANTIGEN RETRIEVAL

Shi et al developed the antigen retrieval technique in 1991 in which high temperature was used to bring out the antigenicity of the tissues which had been masked by formalin fixation. Antigen retrieval is done either by heat induced epitope retrieval or proteolytic epitope retrieval.

HEAT INDUCED

The tissue sections are placed in retrieval solution and subjected to heat for varying periods of time. This breakdowns the protein crosslinks and retrieves antigenicity. ⁹⁵ The heat can be applied using microwave oven, pressure cooker, steamer, autoclave or water bath. The commonly used retrieval solutions are citrate buffer at PH 6, TRIS EDTA at PH 9, EDTA at PH 8.

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28 PROTEOLYTIC EPITOPE RETRIEVAL ⁹⁶

Tissue antigenicity can also be restored using proteases like Proteinase K, trypsin, Chymotrypsin and pepsin. The main disadvantage here is it alters tissue morphology and destroys some epitopes.

TARGET ANTIGEN DETECTION METHODS

After retrieval specific antibodies are added which forms Antigen antibody complex. This can be visualized by Direct and Indirect methods.

DIRECT METHOD

Her labeled antibodies are used which react with antigens in tissue sections. Some of the labels are fluorochrome, horse radish peroxidase and alkaline phosphatase. It is simple and rapid but has low sensitivity.

INDIRECT METHOD

Here, in the first step unlabeled primary antibody is added which binds with the target antigen. Then in the second step, a labeled secondary antibody is added which react with the primary antibody. It is more sensitive and it uses only a small number of secondary antibodies.⁹⁷

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OTHER METHODS OF DETECTING ANDROGEN RECEPTOR AND BETA CATENIN

ANDROGEN RECEPTOR:

Gene expression profiling

BETA CATENIN:

Cell lines and β-catenin knockdown Western blotting

TOPFlash reporter assays

Cell growth, migration and soft agar growth assays Stem cell assays

Quantitative real-time PCR Xenograft transplantation assays

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MATERIALS AND METHODS

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MATERIALS AND METHODS

This study is a descriptive prospective and retrospective study of expression of androgen receptor and beta catenin in triple negative breast carcinomas conducted in the Institute of Pathology, Madras Medical College and Rajiv Gandhi Government General hospital, Chennai during the period between July 2017 to June 2019

Source of data

Triple negative breast carcinoma cases reported in specimens received in the Institute of Pathology, Madras Medical College between July 2017 to June 2019 from the Department of Surgery, Oncology,and Plastic surgery, Government General Hospital were taken.

Inclusion criteria

All mastectomy and trucut biopsy specimens of breast that are histologically and immunohistochemically diagnosed as triple negative breast carcinomas are included.

Exclusion criteria:

 Benign breast lesions

 phyllodes tumors

 Hormone receptor positive breast carcinomas

(45)

31 METHOD OF DATA COLLECTION

Of the total 45 triple negative cases reported during this study period, AR and BETA CATENIN expression was studied for 40 cases. Detailed history of the cases regarding age, sex, menstrual history, side of the breast, type of procedure, history of neo adjuvant therapy, details of gross characteristics such as tumor size, nodal status were obtained for those 40 cases from surgical pathology records.

Formalin fixed tissues were cut, processed and paraffin embedded.

4μm thick sections of the paraffin tissue blocks were cut and stained with eosin and hematoxylin. Slides were collected from slide filing and were reviewed and graded using the Nottingham modification of the Scarff Bloom Richardson Grading system ( Annexure IV ) and they were further evaluated for the presence of necrosis, lymphocytic infiltration lymphovascular invasion and skin infiltration.

The tissue samples were subjected to immunohistochemical analysis of Androgen Receptor and Beta catenin. Slides were evaluated and scoring was given. The results were recorded with photographs.

IMMUNOHISTOCHEMICAL EVALUATION

Immunohistochemical analysis of Androgen receptor and beta catenin were done in Paraffin embedded tissue samples using supersensitive polymer HRP system based on non-biotin polymeric technology.

(46)

32

Table 1: Immunohistochemical markers used in the current study

ANTIGEN VENDOR CLONE DILUTION POSITIVE

ANDROGEN

RECEPTOR PATHNSITU RABBIT

MONOCLONAL

READY TO

USE PROSTATE

BETA

CATENIN PATHNSITU RABBIT

MONOCLONAL

READY TO USE

COLON CARCINOMA

4μm thick sections from selected formalin fixed paraffin embedded tissue samples were transferred onto gelatin coated slides. Heat induced antigen retrieval was done using microwave method.

AR and Beta catenin (PathnSitu) antigens are bound with rabbit monoclonal antibody. Later antigen antibody complex are detected by the addition of secondary antibody conjugated with horse radish peroxidase polymer and Diaminobenzidine substrate.

(47)

33 STATISTICAL ANALYSIS

The Statistical analysis was done using the software IBM Statistical Package for social science version 20. The correlation between AR and Beta catenin expression and different clinicopathological parameters like age group, size, side, grade, lymphnode status, lymphovascular invasion, lymphocytic infiltration, necrosis, skin infiltration were analysed. AR and BETA CATENIN expression was studied and strength of association was calculated by Pearson Chi square test and P value less than 0.05 are considered statistically significant.

(48)

OBSERVATION AND

RESULTS

(49)

34

OBSERVATION AND RESULTS

In a study period of two years from July 2017 to June 2019 a total of 23,348 specimens were received in Institute of Pathology, Madras Medical College for histological examination. Out of which 1668 were breast specimens.Among which 913 are malignant breast tumors cases, 526 cases are benign breast tumours and249 are non-neoplastic lesions and 45 triple negative cases. 40 cases were included in this study.

Among the 40 breast specimens, all were diagnosed malignant following biopsy and all were evaluated for AR and BETA CATENIN. Among the 40 cases, 4were given neo-adjuvant chemotherapy following which modified Radical Mastectomy was done.

The age wise distribution of the cases is given below.

Table 2: Age Wise Distribution Of triple negative breast Cancers

AGE NO. OF CASES PERCENTAGE

30-40 Years 8 20.0

41-50 Years 14 35.0

51-60 Years 12 30.0

Above 60 Years 6 15.0

Total 40 100.0

(50)

35

Highest incidence is seen in age group 41-50 years. The youngest age of presentation in this study is 28 years and older age is 75 years and median age of presentation is 49.5 years.

20%

35%

30%

15%

Chart 1: AGE DISTRIBUTION

30-40 Years 41-50 Years 51-60 Years Above 60 Years

(51)

36

Table 3: Distribution of Histological Subtypes of triple negative breast Cancers

Invasive breast carcinoma constitutes about 95% of triple negative breast cancers in this study and remaining 5% contributed by metaplastic carcinoma.

Chart 2: HISTOLOGICAL TYPE

95%

5%

IBS Nos MC HISTOLOGICAL TYPES NO. OF CASES PERCENTAGE

INVASIVE BREAST

CARCINOMA NOS 38 95.00%

METAPLASTIC

CARCINOMA 2 5.00%

TOTAL 40 100.00%

(52)

37

Table 4: DISTRIBUTION OF SIDE

SIDE NO. OF

CASES PERCENTAGE

LEFT 17 42.50%

RIGHT 23 57.50%

TOTAL 40 100.00%

Of the total 40 cases 17 cases of invasive breast carcinomas were reported in left breast and 23 cases were reported in right breast. Maximum positivity of 57.5% is seen in right breast.

Chart 3 : SIDE OF INVOLVEMENT

43%

57%

0%

10%

20%

30%

40%

50%

60%

Left Right

(53)

38

Table 5 : DISTRIBUTION OF SIZE

SIZE NO. OF CASES PERCENTAGE

<2 cm 1 2.50%

2-5 cm 24 60.00%

>5 cm 4 10.00%

TRU CUT 11 27.50%

TOTAL 40 100.00%

Out of 40 cases 11 cases were trucut biopsies. 1 case (2.5%) had tumor less than 2 cm in size, 24cases (60%) were of 2 to 5 cm in size and 4 cases (10%) were more than 5cm in size

.

Chart 4 : DISTRIBUTION OF SIZE

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

<2 cm 2-5 cm >5 cm 3.45%

82.76%

13.79%

PERCENTAGE

(54)

39

Table 6: DISTRIBUTION OF HISTOLOGICAL GRADE

GRADE NO. OF CASES PERCENTAGE

I 8 20%

II 22 55%

III 10 25%

Total 40 100.0

The breast cancers in the study sample which were graded according to Modified Scarff Bloom Richardson grading system out of which 8 cases(20.00 %) were in grade I, 22 Cases (55.00 %) were in grade II and 10 cases (25.00%) were in grade III.

Chart 5: DISTRIBUTION OF HISTOLOGICAL GRADE

0%

10%

20%

30%

40%

50%

60%

I II III

20%

55%

25%

(55)

40

Table 7: DISTRIBUTION OF STAGE

STAGES NO. OF CASES PERCENTAGE

STAGE I 6 20.69%

STAGE II 18 62.07%

STAGE III 5 17.24%

TOTAL 29 100.00%

Tumours are staged as per TNM classification and majority are in stageII contributing 62.07%. 11 cases were trucut biopsies, hence staging is not applicable.out of 29 cases 20.69% are in stage I,62.07% are in stage IIand 17.24%

are in stageIII.

Chart 6: DISTRIBUTION OF STAGE

20.69%

62.07%

17.24%

STAGE I STAGE II STAGE III

(56)

41

Table 8: DISTRIBUTION OF LYMPHOVASCULAR INVASION LYMPHOVASCULAR

INVASION NO. OF CASES PERCENTAGE

PRESENT 12 30.00%

ABSENT 28 70.00%

TOTAL 40 100.00%

Lymphovascular invasion is seen in 12 cases of invasive breast carcinoma Nos which forms 30 % of the total cases and remaining 28 cases show no lymphovascular invasion.

Chart 7: LYMPHOVASCULAR INVASION

70%

30%

A P

(57)

42

Table 9: DISTRIBUTION OF LYMPHOCYTIC RESPONSE LYMPHOCYTIC

RESPONSE NO. OF CASES PERCENTAGE

PRESENT 9 22.50%

ABSENT 31 77.50%

TOTAL 40 100.00%

Lymphocytic response is seen in 9 cases which accounts for 22.5% and remaining 31 cases show no lymphocytic response which accounts for 77.50 %.

Chart 8: LYMPHOCYTIC RESPONSE

0%

10%

20%

30%

40%

50%

60%

70%

80%

ABSENT PRESENT

78%

22%

(58)

43

Table 10: DISTRIBUTION OF SKIN INVOLVEMENT SKIN INVOLVEMENT NO. OF CASES PERCENTAGE

PRESENT 1 2.50%

ABSENT 39 97.50%

TOTAL 40 100.00%

Skin involvement is seen in only one case out of total cases which accounts for 2.5% of cases.

Chart 9: SKIN INVOLVEMENT 2.50%

97.50%

PRESENT ABSENT

(59)

44

Table 11: DISTRIBUTION OF LYMPH NODE METASTASIS LYMPNODE METASTASIS NO. OF CASES PERCENTAGE

NEGATIVE 29 72.50%

1-3 POSITIVE NODES 8 20.00%

4-10 POSITIVE NODES 2 5.00%

>10 POSITIVE NODES 1 2.50%

TOTAL 40 100.00%

1-3 positive nodes are seen in 8 cases(20%),4-10 positive nodes are seen in 2 cases(5%).>10 positive nodes is seen in one case(2.5%) and 29 cases are negative for nodal metastasis.

Chart 10: LYMPH NODE INVOLVEMENT 72.50%

20.00%

5.00% 2.50%

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

NEGATIVE 1-3 POSITIVE NODES

4-10 POSITIVE NODES

>10 POSITIVE NODES

PERCENTAGE

(60)

45

Table 12: CHEMOTHERAPY

CHEMOTHERAPY NO. OF CASES PERCENT

NOT GIVEN 36 90.0

GIVEN 4 10.0

Total 40 100.0

Chemotherapy is given to four cases after which mastectomy is done .It contributes to 10% of the total cases.

Chart 11: CHEMOTHERAPY

90%

10%

NOT GIVEN GIVEN

(61)

46

Table 13: RECURRENCE:

RECURRENCE NUMBER OF CASES PERCENTAGE

Absent 36 90.0

present 4 10.0

Total 40 100.0

4 cases of invasive breast carcinoma NOS showed recurrence which accounts for 10% of the total cases.

Chart 12: RECURRENCE

90%

10%

Absent Present

(62)

47

Table 14: CORRELATION OF RECURRENCE WITH ANDROGEN RECEPTOR

ANDROGEN_RECEPTOR

Total Negative Positive

Recurrence

Absent Count 29 7 36

% 93.5% 77.8% 90.0%

Present Count 2 2 4

% 6.5% 22.2% 10.0%

Total Count 31 9 40

% 100.0% 100.0% 100.0%

Pearson Chi-Square=1.928 P=0.165

Among the four recurrent cases 2 cases showed AR positivity which accounts for 22.2% which is statistically not significant.

Chart 13: CORRELATION OF RECURRENCE WITH ANDROGEN RECEPTOR

0%

20%

40%

60%

80%

100%

Negative Positive

94%

78%

6%

22%

Absent Present

(63)

48

Table 15: CORRELATION OF RECURRENCE WITH BETA CATENIN BETA_CATENIN

Total Negative Positive

Recurrence

Absent

Count 18 18 36

% 94.7% 85.7% 90.0%

Prese nt

Count 1 3 4

% 5.3% 14.3% 10.0%

Total

Count 19 21 40

% 100.0% 100.0% 100.0%

Beta catenin positivity is seen in 3 cases out of 4 recurrent cases which accounts for 14.3% and the p value is 0.342 which is statistically insignificant.one case showed negative expression of betacatenin.

Chart 14: CORRELATION OF RECURRENCE WITH BETA CATENIN

0%

20%

40%

60%

80%

100%

Negative Positive

95%

86%

5%

14%

Absent Present

(64)

49

Chart15: ASSOCIATED LESIONS

35% of invasive breast carcinomas are associated with other lesions.2.5%

of the cases are associated with ductal carcinoma in situ changes,30% of the case associated with fibrocystic disease of breast and 2.5% cases are associated with intra ductal papillomatosis.

Remaining 65% of the cases are not associated with any lesions.

No Associated Lesion, 65.0%

DCIS, 2.5%

FCD, 30.0%

IDP, 2.5%

Other, 35.0%

(65)

50

RESULTS OF IMMUNOHISTOCHEMISTRY

Table 16:Distribution of Androgen Receptor expression in triple negative breast cancers.

ANDROGEN RECEPTOR EXPRESSION

NO. OF

CASES PERCENTAGE

POSITIVE 9 22.50%

NEGATIVE 31 77.50%

TOTAL 40 100.00%

IHC scoring of AR was done based on staining intensity and proportion of cells exhibiting positivity. shows 9 cases of AR positivity which forms 22.5% of cases.

Chart 16: Distribution of Androgen Receptor expression in triple negative breast cancers

22.50%

77.50%

POSITIVE NEGATIVE

(66)

51

Table 17: Distribution of BETA CATENIN expression in triple negative breast cancers.

BETACATENIN

EXPRESSION NO. OF CASES PERCENTAGE

POSITIVE 21 52.50%

NEGATIVE 19 47.50%

TOTAL 40 100.00%

Beta catenin was expressed by 21 cases which accouns for 52.5% which is concurrent with other studies and 19 cases(47.5%) are negative for beta catenin expression

Chart 17: Distribution of BETA CATENIN expression in triple negative breast cancers

48%

52%

BETA CATENIN

Negative Positive

(67)

52

Table 18: CORRELATION OF ANDROGEN RECEPTOR WITH AGE ANDROGEN

RECEPTOR

Age group

30-40 Years

Count 7 1 8

% 22.6% 11.1% 20.0%

41-50 Years

Count 10 4 14

% 32.3% 44.4% 35.0%

51-60 Years

Count 8 4 12

% 25.8% 44.4% 30.0%

Above 60 Years

Count 6 0 6

% 19.4% 0.0% 15.0%

Total

Count 31 9 40

% 100.0% 100.0% 100.0%

Pearson Chi-Square=3.304 p=0.347

Highest androgen receptor expression is seen in 41-60 years of age which accouns for 44.4% which has a p value of 0.347which is statistically insignificant.

(68)

53

Chart 18: CORRELATION OF ANDROGEN RECEPTOR WITH AGE

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

30-40 Years 41-50 Years 51-60 Years Above 60 Years 23%

32%

26%

19%

12%

44% 44%

0%

Negative Positive

(69)

54

Table 19: CORRELATION OF BETA CATENIN WITH AGE BETA

CATENIN

Total Negative Positive

Age group

30-40 Years

Count 3 5 8

% 15.8% 23.8% 20.0%

41-50 Years

Count 8 6 14

% 42.1% 28.6% 35.0%

51-60 Years

Count 4 8 12

% 21.1% 38.1% 30.0%

Above 60 Years

Count 4 2 6

% 21.1% 9.5% 15.0%

Total

Count 19 21 40

% 100.0% 100.0% 100.0%

Pearson Chi-Square=2.692 p=0.442

41-60 years of age shows highest beta catenin expression which accouns for 29%which has a p value of 0.442 which is statistically insignificant

(70)

55

Chart 19: CORRELATION OF BETA CATENIN WITH AGE

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

30-40 Years 41-50 Years 51-60 Years Above 60 Years 16%

42%

21% 21%

24%

29%

38%

9%

Negative Positive

(71)

56

Table 20: CORRELATION OF TUMOR GRADE WITH ANDROGEN RECEPTOR

ANDROGEN RECEPTOR

GRADE 1

Count 7 1 8

% 22.6% 11.1% 20.0%

2

Count 15 7 22

% 48.4% 77.8% 55.0%

3

Count 9 1 10

% 29.0% 11.1% 25.0%

Total

Count 31 9 40

% 100.0% 100.0% 100.0%

Grade II tumours show maximum AR positivity and accounts for 78%

which parallels with many studies but shows no significant correlation.

(72)

57

Chart 20: CORRELATION OF TUMOR GRADE WITH ANDROGEN RECEPTOR

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Negative Positive

23%

11%

48% 78%

29%

11%

Grade I Grade II Grade III

(73)

58

Table 21: CORRELATION OF TUMOR STAGE WITH ANDROGEN RECEPTOR

ANDROGEN RECEPTOR

Total Negative Positve

STAGE 1

Count 6 0 6

% within

Androgen Receptor 25.0% 0.0% 20.7%

2

Count 14 4 18

% within

3

Count 4 1 5

% within

Total

Count 24 5 29

% within

Androgen receptor expression is seen maximum in stage II tumours which contributes about 80% and has a p value which is insignificant.

(74)

59

Chart 21: CORRELATION OF TUMOR STAGE WITH ANDROGEN RECEPTOR

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Negative Positive

25%

0%

58%

80%

17% 20%

Stage I Stage II Stage III

(75)

60

Table 22: CORRELATION OF TUMOR STAGE WITH BETA CATENIN

BETA CATENIN

GRADE 1

Count 4 4 8

% within

BETA CATENIN 21.1% 19.0% 20.0%

2

Count 10 12 22

% within

BETA CATENIN 52.6% 57.1% 55.0%

3

Count 5 5 10

% within

BETA CATENIN 26.3% 23.8% 25.0%

Total

Count 19 21 40

% within

BETA CATENIN 100.0% 100.0% 100.0%

Beta catenin positivity is seen maximum with stageII tumours contributing 57% which has a p value of 0.960 which is not significant statistically.

(76)

61

Chart 22: CORRELATION OF TUMOR STAGE WITH BETA CATENIN

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Negative Positive

21% 19%

53% 57%

26% 24%

(77)

62

Table 23: CORRELATION OF TUMOR GRADE WITH BETA CATENIN BETA_CATENIN

STAGE

1

Count 4 2 6

% within

BETA CATENIN 28.6% 13.3% 20.7%

2

Count 9 9 18

% within

BETA CATENIN 64.3% 60.0% 62.1%

3

Count 1 4 5

% within

BETA CATENIN 7.1% 26.7% 17.2%

Total

Count 14 15 29

% within

BETA CATENIN 100.0% 100.0% 100.0%

Grade II tumours show 60% positivity to beta catenin expression which is maximum and the p value is 0.296 which is statistically insignificant.

(78)

63

Chart 23: CORRELATION OF TUMOR GRADE WITH BETA CATENIN

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Negative Positive

29%

13%

64%

60%

7%

27%

(79)

64

Table 24: CORRELATION OF TUMOR STAGE WITH ANDROGEN RECEPTOR AND BETA CATENIN

( Both +ve and Both -ve)

AR and BC

Total Both -ve Both +ve

STAGE 1

Count 6 0 6

% within ARANDBC 24.0% 0.0% 20.7%

2

Count 15 3 18

% within ARANDBC 60.0% 75.0% 62.1%

3

Count 4 1 5

% within ARANDBC 16.0% 25.0% 17.2%

Total

Count 25 4 29

% within ARANDBC 100.0% 100.0% 100.0%

Androgen receptor and beta catenin expressions are correlated with stage of the tumour and p value is calculated using chi square test

(80)

65

Chart 24: CORRELATION OF TUMOR STAGE WITH ANDROGEN RECEPTOR AND BETA CATENIN

Both androgen receptor and beta catenin are positive in 75% of stage II cases with a p value of 0.536 which shows no statistical significance.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Both -ve Both +ve

24%

0%

60%

75%

16%

25%

(81)

66

Table 25: CORRELATION OF TUMOR STAGE WITH ANDROGEN RECEPTOR AND BETA CATENIN

( Both +ve , anyone +ve and Both -ve)

AR and BC

Total Both -ve any one +ve Both +ve

STAGE 1

Count 4 2 0 6

% within ar_bc 30.8% 16.7% 0.0% 20.7%

2

Count 8 7 3 18

% within ar_bc 61.5% 58.3% 75.0% 62.1%

3

Count 1 3 1 5

% within ar_bc 7.7% 25.0% 25.0% 17.2%

Total

Count 13 12 4 29

% within ar_bc 100.0% 100.0% 100.0% 100.0%

Both are positive in 75 % of Stage II tumors and anyone marker is positive in 58 % of Stage II tumors and both are negative in 61 % of Stage II tumors with a P value of 0.568 which shows no significant correlation.

(82)

67

Chart 25: CORRELATION OF TUMOR STAGE WITH ANDROGEN RECEPTOR AND BETA CATENIN

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Both -ve any one +ve Both +ve 31%

17%

0%

61%

58%

75%

8%

25% 25%

(83)

68

Table 26: CORRELATION OF TUMOR GRADE WITH ANDROGEN RECEPTOR AND BETA CATENIN

AR and BC

Total Both -ve Both +ve

GRADE 1

Count 8 0 8

% within AR and BC 24.2% 0.0% 20.0%

2

Count 16 6 22

% within AR and BC 48.5% 85.7% 55.0%

3

Count 9 1 10

% within AR and BC 27.3% 14.3% 25.0%

Total

Count 33 7 40

% within AR and BC 100.0% 100.0% 100.0%

Androgen receptor and beta catenin expression is correlated with grade of the tumours and p value calculated. Both shows positivity in 86% of Grade II tumours.

(84)

69

Chart 26: CORRELATION OF TUMOR GRADE WITH ANDROGEN RECEPTOR AND BETA CATENIN

(Both +ve and Both -ve)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Both -ve Both +ve

24%

0%

49%

86%

27%

14%

(85)

70

Table 27: CORRELATION OF TUMOR GRADE WITH ANDROGEN RECEPTOR AND BETA CATENIN

AR, BC

Total Both -ve any one +ve Both +ve

GRADE 1

Count 3 5 0 8

% within

AR, BC 17.6% 31.2% 0.0% 20.0%

2

Count 9 7 6 22

% within

AR, BC 52.9% 43.8% 85.7% 55.0%

3

Count 5 4 1 10

% within

AR, BC 29.4% 25.0% 14.3% 25.0%

Total

Count 17 16 7 40

% within

AR, BC 100.0% 100.0% 100.0% 100.0%

Both are positive in 86 % of Grade II tumors and anyone marker is positive in 44 % of Grade II tumors and both are negative in 53 % Grade II tumors with a P value of 0.343 which shows no significant correlation.

(86)

71

Chart 27: CORRELATION OF TUMOR GRADE WITH ANDROGEN RECEPTOR AND BETA CATENIN

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Both -ve any one +ve Both +ve 18%

31%

0%

53%

44%

86%

29% 25%

14%

(87)

MAY 2020

M.D. PATHOLOGY

BRANCH – III

MAY 2020

CERTIFICATE

DECLARATION

CERIFICATE - II

ABBREVIATION

CONTENTS

INTRODUCTION

INTRODUCTION

AIMS AND OBJECTIVES

AIMS AND OBJECTIVES

REVIEW OF LITERATURE

REVIEW OF LITERATURE

MATERIALS AND METHODS

MATERIALS AND METHODS

OBSERVATION AND

RESULTS

OBSERVATION AND RESULTS

BETA CATENIN

DISCUSSION