Use of algorithm in IHC

Diagnosis of Tumors using Algorithms

Lawrence T. Richards

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

Diagnosis of Tumors using Algorithms

A diagnostic algorithm is a method which utilizes a panel of antibodies intended to solve a diagnostic problem

Many different diagnostic algorithms exist and are available in journals and text books

A diagnostic algorithm is followed by a selective markers for tumor sub classification.

The panel of antibodies selected should be based on the morphological appearance of the tissue and the patient’s clinical history provided by the physician.

Special stain vs IHC MUC-1 PAS MUC-1(IHC) PAS (Special Stain) AdenoCarcinoma (GI)

Markers and Histogenesis

HISTOGENESIS MARKERS

Mesenchymal Vimentin

Epithelial Cytokeratin, EMA

Smooth Muscle Desmin, HHF35, SmActin

Skeletal Muscle Myoglobin

Fibrohistiocyte CD68, Factor XIIIa

Nerve Sheath Leu7, Glial fibrillary acidic protein

Melanocyte HMB 45

Neuronal Neurofilament

Endothelial, perivascular Factor VIII,CD34,CD31

Hematopoitic LCA,CD3,CD20,Ki-1

Neuroendocrine NSE,Chromogranin,Synaptophysin

Ewing’s sarcoma/PNET MIC-2(O-13)

Source:www.moffitt.usf.edu/pubs/ccj/v5nl/department5/html

Basic Flow Chart Sequential First Round (+) ( - ) (+) (+) (+) ( - ) ( - ) ( - ) Second round

Use of Panel of Antibodies Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 + (-) + (-) + + Tumor-1 (-) + (-) + (-) (-) Tumor-2

Differential Application

Tumor 1 Tumor 2

Ab1 + Ab1 –

Ab2 - Ab2 +

Diagnostic Algorithm for unknown Tumor Type Source adopted from: DAKO training manual Positive Positive Negative Negative Vimentin Positive Negative Negative Negative Desmin Negative Positive Negative Negative S 100 / HMB45/ MART-1 Tyrosinase Negative Negative Positive Negative LCA For Poorly differentiated malignant tumor differentiation Caution: Variation in cellular pattern can mislead (+) and (-) . Negative Negative Negative Positive Pan Keratin Notes Sarcoma Melanoma Lymphoma Carcinoma

Undifferentiated Malignant Neoplasm Almost always positive Almost always Negative Sometimes positive Sometimes negative Gowen AM.Diagnostic Immunohistochemistry of Solid Tumors: Strategies and Solutions,USCAP;2002 #35 CD43 PD7/2B11 CD 45 HMB 45 Melanoma antigen V9 Vimentin AE1/AE3 Cytokeratin Sarcoma Melanoma Lymphoma Carcinoma Abs/clone Abs to

Carcinoma of Unknown Primary

The hypothesis is that the primary tumor either remains microscopic and escapes clinical detection or disappears after seeding the metastasis.

Antibody Algorithm is used to

Search for primary Site

Rule out Non-Carcinoma

Like lymphoma, melanoma, sarcoma

Identify Sub-Groups for Treatment

Threshold for Positivity * Positive Generally : unequivocal staining of =10% of tumor cells +/- + -/+ (-) : >90% of tumors positive* : 50-90% of tumors positive* : 10-50% of tumors positive* : <10% of tumors positive* ? : Insufficient evidence [ ] : Staining of secondary (non-neoplastic) cell type ( ) : Staining dependant on antibody

Metastatic Adenocarcinoma of unknown origin with site specific markers PSA + (-) (-) (-) (-) (-) (-) (-) (-) + + + + + + + Prostate Lung Stomach/ Pancreas Breast 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%)

Details of Abs used in the Algorithm Dennis JL et al.Clinical Cancer Research;11:3766-3772,2005 HIER 1:500 DAKO Ks20.8 CK20 HIER 1:50 Novocastro SPT249 TTF-1 HIER 1:100 DAKO ER-PR8 PSA HIER 1:20 Novocastro 5B2 MESOTHELIN ENZYMATIC 1:5000 Novocastro POLY LYSOZOME HIER 1:20 Novocastro 23A3 GCDFP-15 HIER 1:50 Novocastro 6F11 ER HIER 1:500 DAKO OV-TL CK7 HIER 1:100 Novocastro AMT28 CDX2 HIER 1:400 Novocastro Ov185:1 CA 125 A R Dilution Source Clone Abs

Algorithm for Tumors of Unknown Origin Gross Cystic Disease Fluid Protein Source:www.clincancerres.aacrjournals.org/content/vol11/issue10 96 72 Stomach and Pancreas CK7 69 65 Stomach and Pancreas Lysozyme 85 85 Ovary and Pancreas Mesothelin 88 88 Ovary and Pancreas CA 125 95 74 Breast and Ovary ER 96 54 Breast GCDFP-15 97 36 Colon,Stomach, Pancreas CK20 91 68 Colon CK20 98 56 Colon and Stomach CDX-2 96 83 Colon CDX-2 98 91 Lung TTF-1 99 100 Prostate PSA % Specificity % Sensitivity Tumor of Marker

Differential Diagnosis Bladder vs Prostate ca Goldstein N.Immunohistochemical Antibody Panels to help Identify the Primary Sites of Various Carcinomas;ASCP Teleconference # 2109 + (-) PSA + (-) PAP (-) + CEA (-) + Cytokeratin 20 (-) + Cytokeratin 7

Mesothelioma or Small Cell Carcinoma ?

Mesothelioma vs Carcinoma Brown RW et al.Multiple-marker immunohistochemical phenotypes distinguishing malignant pleural mesothelioma from pulmonary adenocarcinoma. Hum Pathol.1993;24:347-354 70 0 B72.3 30 90 Calretinin 80 0 Ber-EP4 30 40 Vimentin 65 0 Placental Alkaline Phosphatase 15 0 CEA 85 10 S-100 75 10 Leu-M1 8 100 Thrombomodulin 100 80 EMA 100 100 Cytokeratin Carcinoma % Mesothelioma % Antigen

Cytokeratin phenotype

CK phenotype

CK7 (-) / CK 20 (-)

CK7 (+) / CK 20 (-)

CK7 (-) / CK 20 (+)

CK7 (+) / CK 20 (+)

Tumors

HNC, Liver, Lung (SqCC and SmCC),Prostate, Renal

Biliary and Pancreas, Breast, Cervical, EM, Lung(Ad ca), Ovarian (non-mucinous), thyroid

Colon, Gastric, Markel C ca

Biliary and Pancreas, Ovarian (Mucinous), Urothelial.

Source: Adopted from Dennis JL, Clin Cancer Res 2005

Cytokeratin for Carcinoma Diagnosis

CK5 CK7 CK20

Breast Ca Ductal ★ ● ○

Lung nonsmall Cell ○ ● ○

(non SqCCa)

Lung SqCCa ● ★ ○

Pancreatic Ca ○ ★ ★

Colorectal Ad Ca ○ ○ ●

Mesothelioma ● ● ○

Prostatic Ad Ca ○ ○ ○

Transitional Cell Ca ● ● ●

★ May be Positive ● Positive ○ Negative

Source: A.M.Gown ASCP Course #35

Prostate Ca or Benign ?

Prostate Cancer

EpCam +

ATM +

AMACR +

PSA + / (-)

34 ßE12 (-) almost all

p63 (-) almost all

Prostein +

NKX3.1 +

Benign Prostate

EpCam (-)

ATM (-) / +

AMACR (-)

PSA + / (-)

34 ßE12 +

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 Ca

EMA

CK7

P63

CK5/6

EpCam

CD57

PSA

PAP

NKX3.1

Prostein

+ -

+ -

+/- 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

Ab Cocktails

Breast Ca : ER + GCDFP-15

Melanoma : MART-1+Melan A+Tyrosine +

PNL2

Basal Cell Marker : p63 + 34betaE12 +

CK5/6

Endocrine Ca: Chrom A+NSE +

Synaptophysin

Pan Sarcoma : Vim + Coll IV + CD 99

Endocervical Ad.Ca & Endometrial 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

A, Normal post-menopausal endometrium (H&E, 20x); B, MUC1 immunostain in normal postmenopausal endometrium showing “pure apical” staining pattern (x20); C, normal endocervical glands (H&E, 20x); D, MUC1 immunostain in normal endocervical glands showing A/C staining pattern, (x20). MUC1-Purely Apical vs Apical & Cytoplasmic Khoury T, et al.BMC Clin Pathol.2006;6:1

Characteristics of antibodies used for evaluation.