“ IHC is an Important Complimentary Tool for Diagnosis of Cancer”

Lawrence T. Richards M.S.,H.T(ASCP), QIHC(ASCP)

Consultant, Bio Marker Assay Development, Santa Barbara, California.

For teaching purpose only

“ The Diagnostic Power of any

Immunohistochemical Procedure is

no Greater than the wisdom of the

Pathologist interpreting it.”

Dr.Allen M.Gown

IHC as a compliment to H&EIHC as a compliment to H&E

• Is H&E alone not enough?

• Is Special Stain not enough?

• Is IHC alone enough?

Special Stain Special Stain

Budding yeast GMS Stain and H&E (Blastomycosis).

AFBHP Alcian Yellow-Toluidine Blue

Antibody for Yeast

Special stain vs IHCSpecial stain vs IHCH.pyloriH.pylori

Alcian Yellow-Toluidine Blue H.Pylori antibody

Usefulness of IHC tests.Usefulness of IHC tests.• Poorly differentiated tumors and mixed carcinomas

• Undifferentiated tumors of unknown origin

• Treatment based on sub-type of cancer: Personalized Medicine eg., Breast Ca

• Monitoring progress of cancer (Predictive and Prognosis)

• In Malignant Lymphoma,

• In identifying Carcinoid (Neuroendocrine) tumors

• In Cytologic specimens

ER/PRAbout 75% of all breast cancers are “ER positive.” They grow in response to the

hormone estrogen. About 65% of these are also “PR positive.” They grow in response to another hormone, progesterone.

If your breast cancer’s cells have a significant number of receptors for either estrogen or progesterone, your cancer is considered hormone-receptor positive and likely to

respond to endocrine therapiesBreast cancer tumors that are ER/PR-positive are 60% likely to respond to endocrine

therapy. Tumors that are ER/PR negative are only 5% to 10% likely to respond to endocrine therapy

Her2In about 20% to 25% of breast cancers, the cancer cells make too much of a protein known as HER2/neu. These breast cancers tend to be much more aggressive and

fast-growing.For women with HER2-positive breast cancers, the drug Herceptin has been shown to

dramatically reduce the risk of recurrenceTriple Negative

Some breast cancers -- estimates range between 10% and 17% -- are known as “triple negative” because they lack estrogen and progesterone receptors and do not

overexpress the HER2 protein. The majority of breast cancers associated with the breast cancer gene known as BRCA1 are triple negative.

These cancers generally respond well to adjuvant chemotherapy

http://www.webmd.com/breast-cancer/breast-cancer-types-er-positive-her2-positive

IHC helps in deciding Treatments

Histiogenic Dx of NeoplasmHistiogenic Dx of Neoplasm• ID of proliferation of cells

 A) Expression of cytokeratin AE1/AE3 in lung carcinosarcoma ; B) chromogranin expression in gastric neuroendocrine carcinoma ; C) HMB 45 immunostainning in melanoma .

Epithelium NeuroEndocrine Melanocyte

Metastatic Adenocarcinoma ofMetastatic Adenocarcinoma of unknown originwith site specific markerswith site specific markers

PSA+(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

+

+

+

+

+

+

+

Prostate

Lung

Stomach/PancreasBreast

Colon

Colon

Stomach / Pancreas

Breast

Ovary

Pancreas,(Ovary serous)

Stomach / Pancreas

Breast / Stomach / Pancreas

Source: www.clincancerres.aacrjournals.org/egi/content/full/11/10/3766

TTF-1

GCDFP15

CDX2 / CK20

ER

CA125

Mesothelin

Lysozyme

+

+

+

(-)

(-)

(-)

CDX2

CK 7

Mesothelin

MC5+ (98%)

Endocervical Ad.Ca & Endocervical Ad.Ca & Endometrial Mucinous Ad.CaEndometrial Mucinous Ad.Ca

ECA• MUC-1(-)• ER(-)• PR(-)• P16(+)

EMMA• MUC-1 (+)• ER(+)• PR(+)• P16(-)

Khoury T et al.BMC Clin Path 2006;6:1

Prostate Ca or Benign ?Prostate Ca or Benign ?• Prostate Cancer• EpCam +• ATM +• AMACR +• PSA + / (-)• 34ßE12 (-) almost all• p63 (-) almost all• Prostein +• NKX3.1 +

• Benign Prostate• EpCam (-)• ATM (-) / +• AMACR (-)• PSA + / (-)• 34ßE12 +• p63 +• Prostein +• NKX3.1 +

Hammerich KH et al.Application of Immunohistochemistry to the Genitourinary System(Prostate, Urinary Bladder, Testis, and Kidney)Archives of Pathology and Laboratory Medicine;132:432-440,2007

ATM=ataxia-telangiectasia mutated;AMACR=alpha-methylacyl-CoA racemase;Ep-Cam=epithelial transmembrane glycoprotein

Urothelial Ca vs Prostate CaUrothelial Ca vs Prostate Ca

• EMA• CK7• P63• CK5/6• EpCam• CD57• PSA• PAP• NKX3.1• Prostei

n

+ -+ -+/- 0+/- 0+/- 0

-/+ +/- 0 +

0 +/- 0 + 0 + 0 +

Urothelial ca Prostate ca

Hammerich KH.Archives of Pathology and Laboratory Medicine;132(3):432-440

Urothelial ca

Prostate ca

PrognasticRisk Biomarkers (or screening biomarkers) Describe risk of cancer occurrence or cancer progression because they are implicated in neoplastic progression and include: 1. Genetic predisposition (e.g., BRCA1/2)2. Over expression of genes (e.g., BCR-ABLTyrosin Kinase Inhibitor in CML, HER-2/neu in Br Ca,PTEN, RAS,Colo Rectal Cancer AKTin Pancreatic cancer)4.Environmental factors and lifestyle (e.g., HPV or HBV infection

http://www.esmo.org/content/download/8713/176680/file/The-use-of-Biomarkers-for-Treatment-Sessa-Fasolo.pdf

Undifferentiated TumorsUndifferentiated Tumors Use of IHC to differentiate broad lineage Use of IHC to differentiate broad lineage

Pan CK

AE1/AE3

CD45

(LCA)

HMB45 or S100

VIM

Carcinoma Positive Negative Negative Negative

Melanoma Negative Negative Positive Positive

Sarcoma Negative Negative Negative Positive

Lymphoma Negative Positive Negative Negative

Carcinoma Carcinoma Use of CK7 and CK20Use of CK7 and CK20

CK7 +

CK20 +

CK7 +

CK20 -

CK7 –

CK20 +

CK7 –

CK20 -

Urothelial Ca

Pancreatic Ad Ca

OvarianMucinous Ca

Ad Ca of Bladder

Gastric Ad Ca

Cholangio Ca subset

Breast Ca

EndoMetrial Ad Ca

EndoCervical Ad Ca

Ovarian Cerous Ca

Lung Ad Ca

Cholangio Ca

LungSmCC

Mesothelioma

Thyroid Ca

SCC of Cervix

SalivaryGland tumors

Urothelial Ca subset

Pancreatic and Gastric Ad Ca subset

Colorectal Ad Ca Markel Cell ca

Gastric Ad Ca subset

Prostate Ad Ca

SCC

RCC

HCC

Mesothelioma

AdrenoCortical ca

NonSeminoma

LungSmCC minorSubset

Gastric Ad Ca subset

http://www.pathinformatics.com/department/documents/SusanE%20Lecture.pdf

Immunohistochemistry stains in squamous cell carcinoma and adenocarcinoma of lung. H&E: hematoxylin and eosin; CK: cytokeratin; TTF-1: thyroid transcription factor 1. Squamous carcinomas are typically positive for CK5/6 and P63, and negative for CK7 and TTF-1, with the reverse profile for adenocarcinoma although this case of squamous cell carcinoma demonstrates focal weak staining for CK7.

SqCC AdCa

“Ancillary Testing in Lung Cancer Diagnosis” Dublinski et al.Openi.nlm.nih.gov

Primary and Additional MarkersPrimary and Additional Markers

For teaching purpose only

For teaching purpose only

CK7 and CK20

http://www.pathinformatics.com/department/documents/SusanE%20Lecture.pdf

For teaching purpose only

Tumors from Unknown Primary SiteTumors from Unknown Primary Site

http://www.translational-medicine.com/content/pdf/1479-5876-10-12.pdf

Metastatic AdenoCarcinoma from Unknown Primary

For teaching purpose only

CUP DiagnosisCUP Diagnosis(Cancer of Unknown Primary)(Cancer of Unknown Primary)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631214/figure/f1-gcr1_6p0229/

For teaching purpose only

Cancer PSA/ PSAP

CK-hmw

CK7 CK20

CK17 T -Mod

CEAm CD57 Leu 7

Prostate Adenoca

+few -

-few +

-foc +

-foc +

-6%+

- -foc +

+few-

Urothelial Ca

-

+ few -

+rare -

+/- + few -

+/- +60-

90%

-foc +

Prostate Ca vs Urothelial Ca

http://www.ihcworld.com/_newsletter/2001/focus_mar_2001.pdf

Hepatocellular Colon Biliary Pancreas Ovary/EM

Sarcoma

Hepar + - - - - -

AFP + - - - - -

CEA - + + +/- +/- -

CK7 - - + + + -

CK20 - + ? ? - -

AE1 AE3

+/- + +/- + + -

CA 19-9 - - - + - -

ER - - - - + -(Not for Renal Cell Carcinoma).(?) suggests could be positive 35% to 65% or negative); and +/- dependent on subtype of histology from that organ.

Immunoperoxidase Panel for Liver Lesions

Napsin A + TTF-1Napsin A + TTF-1

Napsin A, an aspartic acid protease whose expression in the lung is regulated by TTF-1, has also shown promise in helping to differentiate primary lung from metastatic adenocarcinomas. While Napsin A expression may also be seen in normal kidney and in a proportion of renal tumors, positivity for both TTF-1 and napsin A is a strong indication that an adenocarcinoma originated from lung .

Examples of common panels of Antibodies Used

Generic T-cell Vs B-Cell : CD3, CD20, CD45

Folicular Lymphoma Vs Hyperplasia: Bcl2, Bcl6, CD3, CD10,CD20

Low Grade B Lymphoma: CD3, CD5, CD10, CD20, CD23, CD43, Bcl2, Bcl6,

MALT Lymphoma: CD3, Cd5, CD20, Bcl2, ISH Kappa and Lambda

Hodgkin’s Lymphoma: CD3, CD15, CD20, CD30, CD45

Myeloma: CD138, ISH Kappa and Lambda

Carcinoma Vs Lymphoma: CD3, CD20, CD45, PanCK

Metastatic Carcinoma: CK7, CK20, TTF-1

GIST: CD117, CD34, S100, Desmin, SMA

Mesothelioma: PanCK, CK5/6, Calret, TTF-1, CEA, CD15

If male add PSA/ if female add BRST2

Antibodies commonly usedAntibodies commonly used• Breast ER, PR, gross cystic fluid protein, CK7,CK20, E-cadherin

• Colon and other GI tractCEA (monoclonal), CK7, CK20

• Germ cell PLAP, -fetoprotein, -HCG, AE1/3

• Hepatocellular Hepar, -fetoprotein, CK7, CK20, AE1 and AE3 (separately)

• Lung TTF-1, CK7, CK20, CEA, Ber-EP4, chromogranin, synaptophysin, S100

• Lymphoma LCA (CD45RB monoclonal), CD3, CD20, CD30, ALK-1, myeloperoxidase, and light chains, Bcl-2

Antibodies commonly used Antibodies commonly used contd.contd.

• Mesothelioma: Calretinin, AE1, AE3• Melanoma: S100 (when spindled cells), HMB-45

(when epithelioid), MART1• Neuroendocrine : Chromogranin, synaptophysin, NSE• Pancreas : AE1/3, CK7, CK20, CA 19-9, CEA, chromogranin,

synaptophysin, -antichymotrypsin, CD10, PR, Ber-EP4

• Prostate :PSA, CK7, CK20• Renal: EMA, CD10, HMB-45,

inhibin- (to exclude adrenal /cortical)• Sarcoma : Vimentin, S100, CD117 (c-Kit), CD34,SMA, myogenin,

CD31, CD68, desmin,CD1a, CD99• Thyroid: TTF-1, thyroglobulin, calcitonin, CEA• Urothelial : CK7, CK20,Uroplakin

ConclusionConclusion

Thank you for giving me an opportunity to share my knowledge with you all.

It is my hope that that this presentation will fulfill the goal of providing the attendees a little insight into the powerful tool of IHC in diagnosis of difficult cases and help in monitoring disease progression in patients and course correction in treatment procedures.

AcknowledgementsAcknowledgements

New York University

-Department of Environmental Medicine

DAKO

-Research and Development

Quest Diagnostics

-Clinical Trials and Bio-Marker Development