CA BREAST MOLECULAR BIOLOGY AND PATHOLOGY

DR SUHAS K R

Introduction • Breast cancer - extremely heterogeneous disease caused by

interactions of both inherited and environmental risk factors • Progressive accumulation of genetic and epigenetic changes

in breast cancer cells• Tumors with similar clinical and pathological presentations

may have different behaviors• Global gene expression profiling (GEP) studies - for

classifying breast cancer into distinct biological classes associated with patient survival, based on gene expression patterns

GENETICS

Genetics • Low penetrance high frequency breast cancer

predisposition genes o FGFR 2, LSP1, TOX3

• Moderate penetrance low frequency breast cancer predisposition genes o CHEK 2 ,BRIP1, ATM , PALB2

• High penetrance low frequency breast cancer predisposition genes o BRCA1, BRCA 2, TP53, PTEN, CDH 1, STK11/LKB1

BRCA -1Breast Cancer 1,Early onset ( Chr.17)

BRCA-2, Breast Cancer 2,Early onset( Chr.13)

p53( Chr.17) CHEK2( Chr. 22)

FUNCTIONS:1. Transcription2. DNA Repair

of double stranded breaks

3. Ubiquitination

4. Transcriptional regulation.

FUNCTIONS:1. Stability of

the human genome

2. DNA double strand break repair.

FUNCTIONS1. Cell cycle

control2. DNA

replication3. DNA repair4. Apoptosis.

FUNCTIONS1. Cell cycle

checkpoint kinase, recognition and repair of DNA damage.

2. Activates BRCA1 and p53 by phosphorylation

Germline point mutations/Deletions of BRCA1 gene Hereditary breast & ovarian cancers.

Mutations 20% Hereditary breast cancer, ovarian cancer, increased cancer risk in male carriers.

Mutations Sporadic breast cancers. Li fraumeni syndrome

Mutations - rare (<5%). Li fraumeni variantIncrease breast cancer risk after radiation exposure

Differential Chemotherapeutic Sensitivity for Breast Tumors With

BRCA• BRCA1 and BRCA2 germline mutations have an important role in

DSB repair of DNA

• Defective BRCA1 or BRCA2 functions and subsequently impair DNA repair capacity

•  Differentially more sensitive to DNA-damaging chemotherapeutic agents

• Resistant to taxanes

PATHOGENESIS – HORMONAL FACTORS

• Hormones breast growth during puberty, menstrual cycles, pregnancy cycles of proliferation cells at risk for DNA damage.

• If premalignant or malignant cells are present, hormones - stimulate their growth + growth of normal epithelial and stromal cells tumour development.

• Metabolites of estrogen mutations / generate DNA-damaging free radicals.

CLASSIFICATION – BREAST CARCINOMA

NON-INVASIVE/IN SITU CARCINOMA

Intraductal carcinoma Lobular carcinoma in

situ

INVASIVE CARCINOMA

Infiltrating ( invasive ) duct carcinoma – NOS

Infiltrating ( invasive ) lobular carcinoma

Medullary carcinoma

Colloid (mucinous) carcinoma Papillary carcinoma Tubular carcinoma Adenoid cystic carcinoma Secretory carcinoma Inflammatory carcinoma Carcinoma with

metaplasia

PAGET’S DISEASE OF THE NIPPLE

DUCTAL CARCINOMA IN SITUMost DCIS detected by

calcifications on mammography/mammographic density - periductal fibrosis surrounding a DCIS/rarely palpable mass/ nipple discharge/incidental finding on a biopsy for another lesion.

Spreads through ducts & lobules extensive lesions entire sector of a breast.

DCIS – involves lobules – acini distorted, unfolded appear as small ducts.

DCIS – 5 Architectural subtypes

CRIBRIFORM

COMEDO CARCINOMA

PAPILLARY

MICROPAPILLARY

SOLID

ComedocarcinomaSolid sheets of pleomorphic

cells with high grade hyperchromatic nuclei.

Areas of central necrosis +nt.

Necrotic cell membranes – calcify clusters/linear & branching microcalcifications on mammography.

Periductal concentric fibrosis & chronic inflammation.

Extensive lesions – palpable as vague nodularity.

Noncomedo DCISMonomorphic cell population –

nuclear grades low to high.CRIBRIFORM DCIS Intra-epithelial spaces –evenly

distributed, regular in shape.

COOKIE CUTTER – LIKE

• SOLID DCIS Completely fills the involved

spaces.

Noncomedo DCISPAPILLARY DCIS Grows into spaces along

fibrovascular cores lack myoepithelial cell layer.

• MICROPAPILLARY DCIS Bulbous protrusions without a

fibrovascular core arranged in complex intraductal patterns.

Calcifications – assoc.with necrosis/form on intraluminal secretions.

PAGET’S DISEASE OF NIPPLE• Malignant cells/PAGET

CELLS Extend from DCIS within ductal system – via lactiferous sinuses nipple skin without crossing the BM.

• Tumour cells – disrupt tight squamous epithelial barrier – ECF seeps out onto nipple surface oozing scaly crust.

• Paget’s cells – detected by nipple Bx/cytological preparation of the exudate.

• Poorly differentiated, ER Negative, HER2/neu overexpression.

• Prognosis – depends on features of underlying Ca.

PAGET’S DISEASE OF NIPPLE

MANAGEMENT AND PROGNOSIS OF DCISMajor risk factors for recurrence:1. Grade2. Size3. Margins

In ER + ve DCIS Post-op. radiation + Tamoxifen recurrence risk – low.

LOBULAR CARCINOMA IN SITUIncidental biopsy finding -no

calcifications /stromal reactions mammographic densities.

Bilateral - 20% to 40% .

Young women.

Loss of expression of E-cadherin(transmembrane cell adhesion protein cohesion of normal breast epithelial cells).

LOBULAR CARCINOMA IN SITU - MORPHOLOGY• Dyscohesive round cells

with oval or round nuclei and small nucleoli. Absence of atypia, pleomorphism, mitoti activity, necrosis.

Involved acini – recognizable as lobules.

Mucin-positive signet-ring cells.

ER and PR +ve.

Invasive Carcinoma, No Special Type (NST; Invasive

Ductal Carcinoma)Majority (70% to 80%).

Gross appearance: Most tumors - firm to hard ,irregular border . Less frequently - well-circumscribed border , softer consistency.

When cut / scraped characteristic grating sound d/t small, central pinpoint foci or streaks of chalky-white elastotic stroma and occasional small foci of calcification.

Invasive Carcinoma – NST- HPE

Features Well diff. Ca Mod. diff.Ca Poorly diff. Ca.Tubule

formationProminent Less,solid

clusters/single infiltrating cells

Ragged nests/solid

sheets of cellsNuclei Small,round,mo

nomorphicGreater nuclear pleomorphism

Nuclei – enlarged,irregul

ar.Mitotic figures Rare Present NumerousProliferation

rate- - High

Tumour necrosis - - Present

INVASIVE LOBULAR CARCINOMAWell-differentiated and

moderately differentiated carcinomas diploid, ER positive, HER2/neu overexpression - rare

Poorly differentiated carcinomas aneuploid, lack hormone receptors, may overexpress HER2/neu.

Different pattern of metastasis than other breast cancers. Metastasis peritoneum ,retroperitoneum, the leptomeninges (carcinoma meningitis), the gastrointestinal tract, ovaries and uterus.

MEDULLARY CARCINOMAMC - 6th decade.

May closely mimic a benign lesion clinically and radiologically/ present as a rapidly growing mass.

MORPHOLOGY : Well – circumscribed,soft,fleshy mass - little desmoplasia more yielding on palpation and cutting. (medulla =>“marrow”).

MEDULLARY CARCINOMA - HPE1. Solid, syncytium-like sheets of

large cells with vesicular, pleomorphic nuclei, prominent nucleoli > 75% of the tumor

2. Frequent mitotic figures; 3. Moderate to marked

lymphoplasmacytic infiltrate surrounding and within the tumor.

4. Pushing (noninfiltrative) border• Poorly differentiated.

MEDULLARY CARCINOMAHigh nuclear grade, aneuploidy,

hormone receptors - nt, HER2/neu overexpression –nt.

Lymph node metastases -

infrequent.

Syncytial growth pattern and pushing borders - d/t overexpression of adhesion molecules intercellular cell adhesion molecule and E-cadherin limit metastatic potential.

MUCINOUS/COLLOID CARCINOMA

Older women (median age 71) grow slowly - many years.

Morphology: Tumor – soft/rubbery . Consistency & appearance of pale gray-blue gelatin. Borders - pushing / circumscribed.

MUCINOUS CARCINOMA - HPETumor cells - arranged in

clusters and small islands within large lakes of mucin.

Mucinous carcinomas diploid, well to moderately differentiated, and ER positive.

Lymph node metastases - uncommon.

Overall prognosis is slightly better.

INVASIVE PAPILLARY & MICROPAPILLARY CARCINOMA

Rare - 1% or fewer of all invasive cancers.

More commonly seen in DCIS.INVASIVE PAPILLARY CA.ER positive.Favorable prognosis. INVASIVE MICROPAPILLARY

CA.ER negative,HER2 positive. Lymph node metastases - very

commonPrognosis is poor.

METAPLASTIC CARCINOMA Includes a variety of rare types of

breast cancer (<1% of all cases) matrix-producing carcinomas, squamous cell carcinomas, and carcinomas with a prominent spindle cell component.

ER-PR-HER2/neu “triple negative”.

Lymph node metastases - infrequent.

Prognosis - poor.

TUBULAR CARCINOMA• Uncommon.

• Morphology: Well-formed tubules + nt, myoepithelial cell layer, BM - nt tumor cells in direct contact with the stroma. Apocrine snouts - typical.Calcifications - within the lumens.

• > 95% of all tubular carcinomas - diploid, ER + ve,HER2/neu –ve .

• Well differentiated. Excellent prognosis.

INFLAMMATORY CARCINOMATumors swollen,

erythematous breast - caused by extensive invasion and obstruction of dermal lymphatics by tumor cells.

Underlying carcinoma - diffusely infiltrative - does not form a discrete palpable mass confusion with true inflammatory conditions a delay in diagnosis.

Many patients metastases at diagnosis / recur rapidly.

Overall prognosis poor.

Disease patterns in ER +ve

• Have better outcomes

• Potential for recurrence over a long period (half recurrences in between 6 to 15 years )

• Better survival even in a recurrence or a metastatic setting

• predilection for osseous metastasis – women who relapse after a decade do so in bones in majority of cases

Impact of histology • Invasive ductal vs invasive lobular • Invasive lobular associated with older age at presentation ,

larger tumors of lower grade with less LVI • ILC more likely to be bilateral and multicentric and

exclusively ER PR positive and HER 2 negative• ILC tend to be mammographically occult • ILC more likely to involve bone, ovary and the body cavities • Rare sites like skin, adrenal, GB, pancreas more often with ILC Loss of

CDH

Impact of histolgy

HER2/neu• Human Epidermal growth factor Receptor 2

• Member of ErbB protein family.

• HER2 is a cell membrane surface-bound receptor tyrosine kinase - normally involved in signal transduction pathways cell growth and differentiation.

• Approximately 30 % of breast cancers amplification of the HER2/neu gene/ overexpression of its protein product.

HER 2 status • More likely to be detected symptomatically • Younger age, high nuclear grade, more LN and negative

hormone status • Inferior OS • Following BCS locoregional recurrence rates are higher and

re excision rates are higher • Distant mets to liver and lungs • CNS acts as a sanctuary site present in more than 50 %

with mets

Triple negative status • 15% of the breast cancers • Relatively younger population • Larger tumors, mostly grade 3, aggressive phenotype,

less likely to have lymph nodes • Overall poorer prognosis • OS from diagnosis of mets - 7- 12 months • OS from diagnosis of CNS disease - 4 -5 months

Breast cancer subtypes

ER status

Negative

Basal like

Normal like

Positive

Luminal type

Luminal like • ER-positive group - relatively high expression of many

genes expressed by breast luminal cells (ER-responsive genes, luminal cytokeratins and other luminal associated markers)

• Luminal A - 50- 60 % of all breast types

• low histological grade, low degree of nuclear pleomorphism, low mitotic activity (low Ki 67)and include special histological types (i.e., tubular, invasive cribriform, mucinous and lobular) with good prognosis.

• Luminal B 15- 20 % - • increased expression of proliferation-related genes

FGFR1, PI3K

• ER-positive, HER2-negativeand Ki 67 high or ER and HER-2 positive tumors

• more aggressive phenotype, higher histological grade, proliferative index and a worse prognosis

• Ki 67 cut off 14% differentiates the two

Basal like • 8- 37% of all breast cancers • Most of these tumors are infiltrating ductal tumors with

solid growth pattern, aggressive clinical behavior and high rate of metastasis to the brain and lung

• Express high levels of basal myoepithelial markers, such as CK5, CK 14, CK 17 and laminin, triple-negative.

• They also overexpress P-cadherin, fascin, caveolins 1 and 2, alphabeta crystallin and epidermal growth factor receptor (EGFR).

Basal like • triple-negative and basal-like are not completely

synonymous• frequent mutations in the tumor protein 53 (TP53)

gene, retinoblastoma (Rb)

• constitute approximately three quarters of breast

cancer 1 (BRCA1) gene related breast cancers.

Normal breast-like• 5%-10% of all breast carcinomas.• clinical significance remains undetermined• negative for CK5 and EGFR otherwise similar to basal

type on expression of various markers • intermediate prognosis between luminal and basal-like

cancers and usually do not respond to neoadjuvant chemotherapy

TNBC status• Higher loco regional recurrence rates • Significant excess visceral involvement – pulmonary

and CNS • metastatic TNBC - 33- 46 % CNS • Rarely there is a recurrence after 5 years compared to

other subtypes with recurrence even at 17 years • Triple-negative breast cancer is highly responsive to

primary anthracycline and anthracycline/taxane chemotherapy

• A more profound initial response to chemotherapy compared with other phenotypes despite poorer overall survival

CLINICAL GENE EXPRESSION BASEDASSAYS

• PAM 50 –

• a 50 gene expression assay based on microarray and quantitative real time (qRT)-PCR

• provide a risk of relapse score that predicts relapse-free survival for node-negative breast cancer patients who had not received adjuvant systemic therapy

MammaPrint• a microarray based gene expression profiling

assay• The genes that comprise the MammaPrint assay

are proliferation genes and genes associated with invasion and angiogenesis

• 70-gene signature• Stage Ⅰ/Ⅱ, 5 cm, ER (+), Node (-)/[1-3 Node (+)]• FDA approved - Yes

Oncotype DX• most widely used prognostic and predictive• 21 gene qRT-PCR based assay• hormone receptor positive, node-negative breast cancer• 21 selected genes essentially related to proliferation, ERand

HER2 signaling • absolute clinical benefit of adjuvant chemotherapy in lymph

node negative (N-) breast cancer is modest, estimated absolute benefit of 4% in terms of 10-year distant recurrence

• The Trial Assigning IndividuaLized Options for Treatment (Rx)

• GENOMIC GRADE INDEX

• MapquantDx is defines the tumoral histological grade by gene expression features, used to assign a grade index to ER-positive breast cancers in attempt to refine their molecular classification.

• to classify grade 2 tumors into low and high genomic grades

• BREAST CANCER INDEX• the likelihood of distant recurrences in patients diagnosed with

ER-positive, node-negative breast cancer.

THERAPEUTIC TARGETS

Therapeutic targets • Estrogen signalling - therapeutic success story • SERMs, aromatose inhibitors and ovarian ablation • Highly effective and have a made a significant impact

on breast cancer mortality and morbidity • Oncotype DX - adds additional insight

Growth factor receptor pathways

• HER2 (EGFR 2 or Erb2 ) - • HER2 amplification is associated with deregulation of

G1/S phase cell cycle control via up-regulation of cyclins D1, E, and cdk6, as well as p27 degradation

Trastuzumab- • disrupts heterodimeric interaction of HER2 with other

EGFR family members• modulate host immunity, activating natural killer cells

involved in antibody-dependent cellular cytotoxicity• decrease tumor-associated microvessel density

• Lapatinib - inhibits tyrosine phosphorylation of both EGFR and HER 2 which in turn inhibits the activation of proproliferative kinases ERK1/2 and AKT

• IGF – 1R - primary response mediator for IGF

• PI3-K Pathway – central signalling pathway downstream of tyrosine kinases and regulates cell growth and proliferation

Rapamycin - m TOR inhibitors

Raf inhibitor - sorafenib

Angiogenesis • VEGFR 2 mediates most of the functions• Bevacizumab - humanized monoclonal antibody • First line metastatic setting Multi targeted agents Sunitinib - VEGFR, PDGFR and c- kit Sorafenib - VEGFR, RAF kinase

Summary • The histological appearance of the tumors may not be

sufficient to establish the underlying complex genetic alterations and the biological events involved in cancer development and progression

• Defining more detailed biological characteristics to improve patient risk stratification and to ensure the highest chance of benefit and the least toxicity from a specific treatment modality

Thank you