Diagnostic Immunohistochemistry of the Breast...Diagnostic Immunohistochemistry of the Breast Jan Klos MD Department of Pathology Stavanger University Hospital, Stavanger, Norway Conflict

Diagnostic Immunohistochemistry

of the Breast

Jan Klos MD

Department of Pathology

Stavanger University Hospital,

Stavanger, Norway

Conflict of interests

NONE

Breast carcinoma, the most common malignant

tumor in females, is a heterogeneous disease

with a substantial variation in morphology,

immunophenotype and biologic behavior.

Around 5% patients have hereditary background (most

often BRCA1 or BRCA2 mutations)

5 6/15/2019 5

Anatomy and histology of the breast

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Morphologic spectrum of breast carcinoma

Content of the lecture

• Stromal invasion or not?

• Myoepithelial proliferations in breast

• Ductal vs. lobular carcinoma

• Cytokeratins in carcinoma and intraductal

proliferations

• Markers of the breast origin

• Neuroendocrine breast carcinomas

• Prognostic and predictive markers

• Triple negative breast carcinoma

• Future directions

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proliferations






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Disruption or absence of basal membrane and

related structures differentiates between

invasive and non-invasive growth of carcinoma

Invasive vs. in situ carcinoma

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Basal cell markers

• Smooth muscle actin

• Calponin

• Smooth muscle myosin heavy chain (SMMHC)

• P63 – p40 (ΔNp63)

• Podoplanin (D2-40) (possible pitfall - positive endothelial cells in lymphatics)

• CD10

• SOX10

• CK5 (mAb clone XM26, and the rmAbs clones BSR55, EP24 and SP27)

CK5/6 (clone SP53)

• CK14

• The mAb clone 34BE12 should not be used as a general marker

for CK-HMW due to cross-reaction with CK-LMW !!!

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Actin - α-Smooth Muscle Actin

• Cytoplasmic microfilaments important for cell shape, motility, contractility and growth bound with intermediary filaments. Several different chain-types identified but only α-smooth muscle actin has diagnostic application.

• Demonstrated in smooth and striated muscles, myoepithelium, myofibroblasts, vessels, granulosa cells and tumours differentiating these directions. Negative staining is found in carcinomas, some sarcomas, schwannomas, neurofibroma. Most of melanomas are negative, but some desmoplastic/spindle cell variants are reported positive

• Application: demonstration of myoepithelial cells and subtyping of tumours

Clone 1A4 is most widely used (platform dependant), but BS66 and rmAb clone EP188 could be used to obtain the optimal staining.

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Breast - α-Smooth Muscle Actin

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Pitfall: SMA clone 1A4 staining

SMA p63

Smooth Muscle Myosin Heavy

Chain (SMMHC)

• Together with light chains forms a myosin complex in smooth

muscle cells and myoepithelial cells. Present only in limited

amount in myofibroblasts.

• Used as sensitive and fairly specific marker of smooth muscle and

myoepithelial differentiation in tumours as well as a sensitive

marker of normal myoepithelial cells.

The mAb clones SMMS-1 and S131 can give optimal results

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Smooth Muscle Myosin Heavy

Chain (SMMHC)

p63 - p40 (ΔNp63)

• Plays an important role in regulating epithelial development and

differentiation (transcription factor for squamous and urothelial

differentiation) and participates also in regulation of p53 effect

• Restricted to certain cells and tissues: squamous epithelium,

urothelium, basal cells of outer cell layer in prostate acini and ducts,

pseudostratified columnar epithelium, reserve cells of endocervix,

pancreatic ducts, myoepithelium, salivary glands, oocytes (but not

testicular germ cells), …

Negative in mesenchymal cells, lymphoid cells (except P63positivity

in GCC cells), neural and normal neuroendocrine tissues.

• P40 considered more specific than P63.

Clones mAb 4A4, DAK-p63 and rmAb DBR16.1 may give optimal staining

for P63 and mAb clones BC28 and ZR8 may do the same for P40.

P63/P40

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SMMHC + p63

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DCIS - myoepithelial cells

Decreased expression of one or more myoepithelial

cell markers found in 85 out of 101 cases of DCIS!

Hilson JB et al. Am J Surg Pathol 2009;33:227-232

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Antibody % cases with lost reactivity

SMMHC 77%

CD10 34%

CK5/6 30%

Calponin 17%

P63 13%

P75 4%

α-SMA 1%

Myoepithelial cells in DCIS

Actin p63 6/15/2019 21

Phenotypic alterations of myoepithelial cells

Actin

p63

Adenoma of the nipple

Infiltrating or not?

P63 ACTIN SMA

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Actin 1A4 Actin 1A4

CK 5/6 P63

Take home message

Phenotypic alteration of myoepithelial cells may cause diagnostic

problems especially in cases of:

• Microglandular adenosis (Actin-/p63-/S-100+)

• Adenoma of the nipple (Actin-/p63+…)

• Some ductal structures in radial scar (Actin-/p63+/-…)

• DCIS (Actin-/+/p63+/-…)

Use more than one myoepithelial marker especially in doubtful cases

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Infiltrating carcinoma with in situ like pattern exists! Be aware!






proliferations






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Myoepithelial cells in infiltrating

carcinoma

• May represent remnants of normal structures.

• Some infiltrating carcinomas (i.e. adenoid cystic carcinoma) show

presence of myoepithelial cells at the periphery of infiltrating foci.

• Myoepithelial differentiation in infiltrating breast carcinomas is

commonest among triple negative (ER-/PgR-/Her2-) cases.

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Actin

34BE12

p63

Adenoid Cystic Carcinoma

Primarily diagnosed as fibromatosis!

Fibromatosis???

Actin - SMA CK-7

Ki-67

Fibromatosis-like breast carcinoma

with myoepithelial phenotype

PAN-CK (AE1/AE3+ 5D3) CK5/6 P63

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Myoepithelial cell proliferations

and tumours in breast

• Myoepitheliosis

Intraductal

Periductal

• Adenomyoepithelial adenosis

• Adenomyoepithelioma

Benign

With malignant change (in one or both components)

• Malignant myoepithelioma/myoepithelial carcinoma

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Adenomyoepithelial adenosis

CK5/6, CK14, CKHMW p63

S-100 Actin

Malignant adenomyoepithelioma

of the breast

• Rare as malignant variant

• Both components should fulfill criteria of

malignancy and show characteristic immunoprofile

for epithelial and myoepithelial differentiation

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Breast tumour in 59 yrs old woman

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Vimentin

CK5/6

CK14

S-100 p63 6/15/2019 38

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Vimentin

Actin

PanCK (5D3+LP34)

S-100

CK5/6

CK14

P63 tumor P63 normal breast

PanCK (5D3+LP34) tumor PanCK (5D3+LP34) normal breast




• Ductal NOS vs. lobular carcinoma


proliferations






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More than 30 histological subtypes of

infiltrating breast carcinoma:

• Ductal NOS (mixed, pleomorphic, …)

• Lobular (with subtypes)

• Tubular

• Cribriform

• Medullary features

• Mucoepidermoid

• Polymorphous

• Mucinous and signet-ring-cell

• Neuroendocrine

• Solid papillary

• Papillary invasive

• Micropapillary

• Metaplastic (with different subtypes)

Subtyping of breast carcinomas

• Apocrine • Lipid rich • Secretory • Oncocytic • Adenoid cystic

• Acinic cell

• Glycogen rich

• Sebaceous

• Inflammatory

• …..

Ductal NOS vs. lobular carcinoma

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Ductal NOS vs. lobular carcinoma

• E-Cadherin (CD325) is a critical regulator of epithelial junction formation.

It interacts with the cytoskeleton through a number of associated proteins.

The internal domain binds with alpha, beta, gamma, and p120 catenins to

anchor the E-Cadherin complex to the actin cytoskeleton of the cell.

E-Cadherin is expressed in virtually all normal epithelial cells with the

exception of adrenocortical cells. The expression in liver cells is weaker

than in most other epithelia.

The mouse monoclonal Ab (mAb) clones NCH-38, 36, 36B5, 4A2C7, DBM

15.49, ECH-6, HECD-1 and GM016 can be used to obtain optimal staining

• P120 catenin is a part of a cadherin complex. Gives distinct, diffuse

cytoplasmic staining in the cases of lobular carcinoma, but membranous

staining in the cases of ductal carcinoma. Useful in differential diagnosis of

lobular vs. ductal lesions. Ref. D.Dabbs et al. Am J Surg Pathol. 2007;31:427-37

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Lobular vs. ductal carcinoma

From Dabbs J.D. et al. Am J Surg Pathol 2007, 31; 427-37

E-Cadherin and p120

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DCIS vs. LCIS

E-Cadherin

E-Cadherin

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10% lobular ca are E-Cadherin+

10-15% ductal ca are E-Cadherin-

Infiltrating lobular carcinoma with

E-Cadherin negative DCIS 6/15/2019 Infiltrating lobular carcinoma

with E-Cadherin positive ducts

E-Cadherin

E-Cadherin in lobular carcinoma

49

“Collision” carcinoma

E-Cadherin






proliferations






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Cytokeratins in infiltrating carcinoma

• Majority of infiltrating carcinomas are positive for Low Molecular Weight Cytokeratins (CK19 ) while non malignant lesions are negative.

• CK7+/CK20- is typical for >95% breast carcinomas

• Lobular carcinoma shows consistent positivity for CKHMW (clone 34β12E), but the presence of High Molecular Weight Cytokeratins is not confirmed! (cross reactivity probably with denaturated epitope on CK19?)

• Some ductal carcinomas are positive for CKHMW (clone 34β12E) and may show presence of CK14 or CK5/6

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Cytokeratins in infiltrating carcinoma

CK19 CK5/6

>95% of breast carcinoma is CK7+/CK20-

CK7 54

55

High molecular weight cytokeratins

in hyperplasia and in situ lesions

IDH ADH DCIS LCIS

CKHMW

(34ßE12 !)

Positive

+++

90-100%

Negative

-/+

80-100%

Negative

-/+

81-100%

Positive

+++

80-100%

CK 5/6

Positive

+++

88-100%

Negative

-/+

80-92%

Negative

-

96-100%

Negative

-/+

83-100%

CK19

(LMWCK)

Negative Variable

+/-

Positive

+

Positive

+

Am J Surg Pathol 2004;28:1076-1091

55

IDH vs. ADH vs. DCIS

CK5/6 CK19

DCIS - Cancerisation of the lobules

CK5/6






proliferations






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Primary breast tumor?

• GCDFP15

• Mammaglobin

• GATA 3

• Hormone receptor status is not a reliable marker of

the breast origin!

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ER and PgR in tumours

• Breast carcinoma +/–

• Ovarian (non-muc.) +/–

• Endometrial +/–

• Cervical –/+

• Lung –/+

• Kidney –/+

• Neuroendocrine –/+

• .......

Sica G et al. APLM 2008,132;1889-95

Results vary with different antibody clones usually

lowest for 1D5, moderate for 6F11 and highest for SP1

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Gross Cystic Disease Fluid Protein 15

• Prolactin induced glycoprotein, secreted by cells of mammary,

sweat and salivary glands. The antibody stains 30-60% of breast

carcinomas, particularly lobular and apocrine type.

• Highly sensitive for mammary and extrammary Pagets disease.

• Considered more specific, but less sensitive than Mammaglobin

marker for breast carcinoma.

• Few positive cases of lung, ovary and renal cell carcinomas are

reported.

• The mAb clones 23A3, D6 and the rmAb clone EP1582Y can give

optimal results.

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GCDFP15

Normal breast

Lobular ca in omentum

Ductal ca in bone 62

Mammaglobin

• Mammary-specific secretory protein member of the uteroglobin

family. Gives positive staining (60-85%) in mammary, sweat and

salivary glands and their tumours.

• Other positive tumours are endometrioid carcinoma (20-85%),

melanoma (5-10%), endocerivcal carcinoma in situ (40-50%).

Single positive cases of adenocarcinoma of pancreas, lung, ovary,

thyroid, stomach are reported

• Considered more sensitive but less specific than GCDFP15

marker for breast carcinoma

• The mAb clone 304-1A5 and the rmAb clone 31A5 can give

optimal results.

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Mammaglobin

GATA3

Transcription factor important in the

differentiation of breast epithelia,

urothelia and subset of T- lymphocytes.

Expressed in many different tissues

with very high frequency in normal

breast and breast carcinomas: 100% lobular breast carcinoma, yolk sac tumor and

choriocarcinoma;

~90% ER+ or HER2+ ductal breast carcinoma (~60%

triple negative breast cancers), skin adnexal tumors

~80% urothelial carcinoma and squamous cell ca of skin,

~50% chromophobe renal cell carcinoma

~40% salivary gland carcinomas and pancreas adca

~30% malignant mesothelioma

squamous cell carcinoma of uterine cervix (~30%),

larynx (~20%) and lung (~10%)….

The mAb clone L50-823 and the rmAb

clone EP368 can give optimal results.

Lobular breast carcinoma

Ductal breast carcinoma






proliferations






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Metastatic neuroendocrine ca in liver

67 Chromogranin A

CK7

ER

GCDFP-15

Carcinomas with neuroendocrine

features

• Normal breast has no endocrine cells!

• Neuroendocrine carcinomas are rare (less than 1%)

• Well differentiated neuroendocrine tumour

• Poorly differentiated neuroendocrine tumour

• Foci of neuroendocrine differentiation in invasive carcinomas can

be seen in ~ 30%

• Mucinous carcinoma, solid papillary carcinoma are diffuse positive

for neuroendocrine markers > 50% cases

• Well differentiated neuroendocrine carcinomas usually express

hormone receptors, but poorly differentiated only in ~50%

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Neuroendocrine differntiation

Synaptophysin in “ordinary“ DCIS

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Small cell carcinoma of the breast

Chromogranin A Ki-67 Synaptophysin ER PgR

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Solid papillary carcinoma

Chromogranin A ER/PgR Synaptophysin KI-67

AB/PASD

72

Neuroendocrine differentiation is quite common

in mucinous breast carcinomas

Chromogranin A

72






proliferations

• Neuroendocrine carcinoma





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Prognostic and predictive factors

Prognostic • Lymph node status

• Tumour size

• Tumour grade

• Hormone receptor status

• Her2 status in LN+

• Ki-67 fraction

Predictive • ER status

ER- strong negative power

ER+ less positive predictive power

• Her2 status

Her2+ strong negative power

• Tumour grade

High grade predicts response to

chemotherapy

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Estrogen receptor

ER (ESR) • Estrogen binding nucleoprotein

present in ERα and ERβ forms acting as transcription factor

• Expressed in different cells and tissues in both sexes also outside reproductive organs

• ERα induces cell proliferation in target tissues

• Expressions of ERα correlates with response to antioestrogen therapy in breast carcinoma

• Clones: rmb SP1 and EP1 give the best performance

• Progesterone binding nucleoprotein induced by Estrogen/ER complex, acting as transcription regulator

• Expressed mainly in female reproductive organs but also other tissues

• Contributes to cell proliferation and differentiation

• Weaker correlation than ER with response to hormonal therapy in breast carcinoma

• Clones: mAb clones 16, PgR 636, PgR 1294, and rmAb clone 1E2 all perform well

Progesterone receptor

PgR (PGR)

Estrogen and Progesterone Receptors

PgR ER

PgR ER in sentinel node

Scoring hormone receptors

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Her2/neu

• Transmembrane glycoprotein encoded on chr 17

• Involved in cell growth and proliferation

• Overexpressed as a result of gene amplification in 15-20% of breast carcinomas mostly of ductal type and some other tumors

• Expression correlates often with ER-/PgR- phenotype and aggressive clinical course

• Target molecule for humanized antibody Trastuzumab

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Her2 status

Score 0 Score 1+ Score 2+ Score 3+

Herceptin Therapy (15-20%)

Non-amplified

Am

plifie

d

35% 35% 15% 15%

80% 20% ~5% FISH + 90% FISH+

Response rate: 15-40%, but with

combined therapy 60% or more Courtessy of I. Skaland

Prognosis and proliferation

Ki-67

81

Baak J et al, Error sources in breast cancer proliferation

Treatment of breast carcinoma

• Often combination of different treatment modalities, where status

of hormone receptors (ER and PgR) determined by

immunohistochemistry as well as Her2 status determined by

immunohistochemistry and/or FISH decide about the choice of

therapy.

• Antiestrogen therapy (blocking estrogen receptors or blocking

synthesis of estrogens) is given to the patients with 1% positive

tumor cells

• Anti-HER2 therapy (Trastuzumab, Pertuzumab) is given to

patients overexpressing HER2 protein or showing amplification of

Her2 gene.

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proliferations






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ER-, PgR-, Her-2-

Triple negative breast carcinoma

• Heterogeneous group

• Bad prognosis in majority of cases

• Some effects of PARP-inhibitors and PDL-1 inhibition

Triple negative with bad prognosis

ER- PgR- Her-2- Ki-67 CK5/6

Basal-like carcinomas: ER- / PR- / HER2-

~15% of all breast carcinomas, poorly differentiated, express: CK 5/6, CK17, EGFR (+)

Triple Negative but not basal

Basal but not

triple negative

BRCA 1-2

• Definition by gene expression

• Includes most BRCA1 mutated tumors

• 15-40% are ER+, PR+ or HER2+

• BRCA1-2 mutated tumors

• ~5% of Breast Cancer

• >50% BRCA-1 mutated are basal-like

75% of

triple

negative

with basal

gene

expression

• Definition by IHC

• Includes different histological types

• >90% of TNBC do not have BRCA mutations

“Triple negative” breast carcinoma only partially overlap with “basal-like” breast carcinoma

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Triple negative breast carcinoma

with good prognosis

Adenoid cystic carcinoma

Secretory carcinoma

Acinic cell carcinoma

Low grade mucoepidermoid carcinoma

CK5/6






proliferations






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90

Molecular subtypes and IHC

Hormone

receptors

Her2 Other %

Luminal A ER+++

PgR+/-

- 55

Luminal B ER+/-

PgR+/-

- 15

Her2 + ER-

PgR-

+ 5

Basal like ER-

PgR-

- CK5/6+/-

EGFR+/-

15

Luminal C ER-/+

PgR-/+

+ 10

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Molecular profiling

Molecular Tests Most Commonly Used for Breast Cancer

Current Clinical Practice

• Test Analyte Method Role in Care

ER, PR expression Protein IHC Subclassify tumor

Predict benefit of hormonal therapy

HER2 overexpression Protein IHC Subclassify tumor

• Predict benefit of targeted therapy

HER2 amplification Tumor DNA FISH Subclassify tumor

• Predict benefit of targeted therapy

Gene panel testing Tumor DNA NGS Identify predictive or prognostic mutations

• (actionable mutations)

MammaPrint Tumor RNA Microarray Predict recurrence

• Select patients for chemotherapy

Oncotype DX Tumor RNA RT-PCR Predict recurrence

• Select patients for chemotherapy

Prosigna Tumor RNA Microarray Predict recurrence

• breast cancer assay Select patients for chemotherapy

Germ line testing Nontumor DNA Sanger or NGS Identify genetic predisposition

• Counsel patients and relatives

• Guide screening and treatment

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Future Directions

Next Generation Immunohistochemistry

• Integration of IHC and molecular technologies is natural and will

accelerate resulting in better understanding and better treatment of

tumors.

• IHC being capable to identify proteins which are a result of

molecular alterations will act as a screening test or as a surrogate

of molecular tests, since its less expensive, less time consuming

and widely available.

• The number Companion Diagnostic tests will dramatically

increase following the development of targeted therapy.

• Quality Management of the whole IHC process will be obligatory

for standardization and reproducibility of the results at the intra- and

inter-laboratory level.

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Thank you!

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Documents

Diagnostic Immunohistochemistry of the Breast...Diagnostic Immunohistochemistry of the Breast Jan Klos MD Department of Pathology Stavanger University Hospital, Stavanger, Norway Conflict