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Diagnosis of Diagnosis of TumorsTumors using using

AlgorithmsAlgorithms

Lawrence T. RichardsLawrence T. RichardsM.S.,H.T(ASCP).,QIHC(ASCP).M.S.,H.T(ASCP).,QIHC(ASCP).

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Diagnosis of Tumors Diagnosis of Tumors using Algorithmsusing Algorithms

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

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

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

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

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MUC-1 PAS

MUC-1(IHC) PAS (Special Stain)

AdenoCarcinoma (GI)

Special stain vs IHCSpecial stain vs IHC

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Markers and HistogenesisMarkers and Histogenesis HISTOGENESISHISTOGENESIS MARKERSMARKERS MesenchymalMesenchymal VimentinVimentin EpithelialEpithelial Cytokeratin, EMACytokeratin, EMA Smooth MuscleSmooth Muscle Desmin, HHF35, Desmin, HHF35,

SmActinSmActin Skeletal MuscleSkeletal Muscle MyoglobinMyoglobin FibrohistiocyteFibrohistiocyte CD68, Factor XIIIaCD68, Factor XIIIa Nerve SheathNerve Sheath Leu7,Leu7,Glial fibrillary acidic Glial fibrillary acidic

proteinprotein MelanocyteMelanocyte HMB 45HMB 45 Neuronal Neuronal Neurofilament Neurofilament Endothelial, perivascularEndothelial, perivascular Factor Factor

VIII,CD34,CD31VIII,CD34,CD31 HematopoiticHematopoitic LCA,CD3,CD20,Ki-1LCA,CD3,CD20,Ki-1 NeuroendocrineNeuroendocrine

NSE,Chromogranin,SynaptophysinNSE,Chromogranin,Synaptophysin Ewing’s sarcoma/PNETEwing’s sarcoma/PNET MIC-2(O-13)MIC-2(O-13)

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

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Basic Flow ChartBasic Flow ChartSequentialSequential

First Round

(+) ( - )

(+)

(+)

(+)

( - )

( - )

( - )

Second round

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Use of Panel of Use of Panel of AntibodiesAntibodies

Ab1 Ab1 Ab2Ab2 Ab3Ab3 Ab4Ab4 Ab5 Ab5 Ab6Ab6

+ (-)+ (-) + + (-)(-) + + + Tumor-1+ Tumor-1

(-)(-) ++ (-)(-) ++ (-)(-) (-) Tumor-2(-) Tumor-2

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Differential ApplicationDifferential Application

Tumor 1Tumor 1 Tumor 2Tumor 2 Ab1 +Ab1 + Ab1 –Ab1 – Ab2 -Ab2 - Ab2 +Ab2 +

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Diagnostic AlgorithmDiagnostic Algorithmfor unknown Tumor Typefor unknown Tumor Type

CarcinoCarcinomama

LymphoLymphomama

MelanoMelanomama

SarcomaSarcoma NotesNotes

Pan Pan KeratinKeratin

PositivePositive NegativeNegative NegativeNegative NegativeNegative ForFor

Poorly Poorly differentiatedifferentiated malignant d malignant tumor tumor differentiatiodifferentiationn

Caution:Caution:

Variation in Variation in cellular cellular pattern can pattern can misleadmislead

(+)(+) and and (-)(-)..

LCALCA NegativeNegative PositivePositive NegativeNegative NegativeNegative

S 100 / S 100 / HMB45/HMB45/

MART-1MART-1

TyrosinaseTyrosinase

NegativeNegative NegativeNegative PositivePositive NegativeNegative

DesminDesmin NegativeNegative NegativeNegative NegativeNegative PositivePositive

VimentinVimentin NegativeNegative NegativeNegative PositivePositive PositivePositive

Source adopted from: DAKO training manual

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Undifferentiated Undifferentiated Malignant NeoplasmMalignant Neoplasm

Abs toAbs to Abs/Abs/cloneclone

CarcinoCarcinomama

LymphoLymphomama

MelanoMelanomama

SarcomaSarcoma

CytokerCytokeratinatin

AE1/AE3AE1/AE3

VimentinVimentin V9V9

MelanoMelanoma ma antigenantigen

HMB 45HMB 45

CD 45CD 45 PD7/2B1PD7/2B111

CD43CD43

Almost always positive

Almost always Negative Sometimes positive

Sometimes negative

Gowen AM.Diagnostic Immunohistochemistry of Solid Tumors: Strategies and Solutions,USCAP;2002 #35

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Carcinoma of Carcinoma of Unknown PrimaryUnknown Primary

The hypothesis is that the The hypothesis is that the primary tumor either remains primary tumor either remains

microscopic and escapes clinical microscopic and escapes clinical detection or disappears after detection or disappears after

seeding the metastasis.seeding the metastasis.

Antibody AlgorithmAntibody Algorithm is used to is used toSearch for primary SiteSearch for primary SiteRule out Non-Carcinoma Rule out Non-Carcinoma

Like lymphoma, melanoma, Like lymphoma, melanoma, sarcomasarcoma

Identify Sub-Groups for Identify Sub-Groups for TreatmentTreatment

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: >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

* Positive Generally : unequivocal staining of =10% of tumor cells

 

Threshold for PositivityThreshold for Positivity

+/-

+

-/+

(-)

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Metastatic Adenocarcinoma ofMetastatic Adenocarcinoma of unknown origin

with 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%)

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Details of Abs used in the Details of Abs used in the AlgorithmAlgorithmAbsAbs CloneClone SourceSource DilutionDilution A RA R

CA 125CA 125 Ov185:1Ov185:1 NovocastroNovocastro 1:4001:400 HIERHIER

CDX2CDX2 AMT28AMT28 NovocastroNovocastro 1:1001:100 HIERHIER

CK7CK7 OV-TLOV-TL DAKODAKO 1:5001:500 HIERHIER

CK20CK20 Ks20.8Ks20.8 DAKODAKO 1:5001:500 HIERHIER

ERER 6F116F11 NovocastroNovocastro 1:501:50 HIERHIER

GCDFP-15GCDFP-15 23A323A3 NovocastroNovocastro 1:201:20 HIERHIER

LYSOZOMELYSOZOME POLYPOLY NovocastroNovocastro 1:50001:5000 ENZYMATIENZYMATICC

MESOTHELIMESOTHELINN

5B25B2 NovocastroNovocastro 1:201:20 HIERHIER

PSAPSA ER-PR8ER-PR8 DAKODAKO 1:1001:100 HIERHIER

TTF-1TTF-1 SPT249SPT249 NovocastroNovocastro 1:501:50 HIERHIER

Dennis JL et al.Clinical Cancer Research;11:3766-3772,2005

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Algorithm for Tumors of Algorithm for Tumors of Unknown OriginUnknown Origin

MarkerMarker Tumor ofTumor of % Sensitivity% Sensitivity % Specificity% SpecificityPSAPSA ProstateProstate 100100 9999

TTF-1TTF-1 LungLung 9191 9898

CDX-2CDX-2 ColonColon 8383 9696

CDX-2CDX-2 Colon and Colon and StomachStomach 5656 9898

CK20CK20 ColonColon 6868 9191

CK20CK20Colon,Stomach,Colon,Stomach,

PancreasPancreas 3636 9797

GCDFP-15GCDFP-15 BreastBreast 5454 9696

ERER Breast and OvaryBreast and Ovary 7474 9595

CA 125CA 125 Ovary and Ovary and PancreasPancreas 8888 8888

MesothelinMesothelin Ovary and Ovary and PancreasPancreas 8585 8585

LysozymeLysozyme Stomach and Stomach and PancreasPancreas 6565 6969

CK7CK7 Stomach and Stomach and PancreasPancreas 7272 9696

Gross Cystic Disease Fluid Protein

Source:www.clincancerres.aacrjournals.org/content/vol11/issue10

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Differential DiagnosisDifferential Diagnosis Bladder vs Bladder vs

Prostate ca Prostate ca

Cytokeratin Cytokeratin 77

++ (-)(-)

Cytokeratin Cytokeratin 2020

++ (-)(-)

CEACEA ++ (-)(-)

PAPPAP (-)(-) ++

PSAPSA (-)(-) ++

Goldstein N.Immunohistochemical Antibody Panels to help Identify the Primary Sites of Various Carcinomas;ASCP Teleconference # 2109

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Mesothelioma or

Small Cell Carcinoma ?

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Mesothelioma vs CarcinomaMesothelioma vs CarcinomaAntigenAntigen Mesothelioma %Mesothelioma % Carcinoma %Carcinoma %

CytokeratinCytokeratin 100100 100100

EMAEMA 8080 100100

ThrombomodulinThrombomodulin 100100 88

Leu-M1Leu-M1 1010 7575

S-100S-100 1010 8585

CEACEA 00 1515Placental Alkaline Placental Alkaline PhosphatasePhosphatase

00 6565

VimentinVimentin 4040 3030

Ber-EP4Ber-EP4 00 8080

CalretininCalretinin 9090 3030

B72.3B72.3 00 7070Brown RW et al.Multiple-marker immunohistochemical phenotypes distinguishing malignant Brown RW et al.Multiple-marker immunohistochemical phenotypes distinguishing malignant pleural mesothelioma from pulmonary adenocarcinoma. Hum Pathol.1993;24:347-354pleural mesothelioma from pulmonary adenocarcinoma. Hum Pathol.1993;24:347-354

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Cytokeratin Cytokeratin phenotypephenotype CK phenotypeCK phenotype CK7 (-) / CK 20 CK7 (-) / CK 20

(-)(-)

CK7 (+) / CK 20 CK7 (+) / CK 20 (-)(-)

CK7 (-) / CK 20 CK7 (-) / CK 20 (+)(+)

CK7 (+) / CK 20 CK7 (+) / CK 20 (+)(+)

TumorsTumors HNC, Liver, Lung (SqCC and HNC, Liver, Lung (SqCC and

SmCC),Prostate, RenalSmCC),Prostate, Renal

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

Colon, Gastric, Markel C caColon, Gastric, Markel C ca

Biliary and Pancreas, Ovarian Biliary and Pancreas, Ovarian (Mucinous), Urothelial.(Mucinous), Urothelial.

Source: Adopted from Dennis JL, Clin Cancer Res 2005

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Cytokeratin for Carcinoma Cytokeratin for Carcinoma DiagnosisDiagnosis

CK5CK5 CK7CK7 CK20CK20Breast Ca DuctalBreast Ca Ductal ★★ ● ● ○ ○Lung nonsmall CellLung nonsmall Cell ○ ○ ● ● ○ ○ (non SqCCa) (non SqCCa) Lung SqCCaLung SqCCa ● ● ★ ★ ○ ○Pancreatic Ca Pancreatic Ca ○ ○ ★ ★ ★ ★ Colorectal Ad Ca Colorectal Ad Ca ○ ○ ○ ○ ● ●Mesothelioma Mesothelioma ● ● ● ● ○ ○Prostatic Ad CaProstatic Ad Ca ○ ○ ○ ○ ○ ○ Transitional Cell CaTransitional Cell Ca ● ● ● ● ● ●

★ ★ May be Positive ● Positive ○ NegativeMay be Positive ● Positive ○ Negative

Source: A.M.Gown ASCP Course #35Source: A.M.Gown ASCP Course #35

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Prostate Ca or Prostate Ca or Benign ?Benign ?

Prostate Prostate CancerCancer EpCam +EpCam + ATM +ATM + AMACR +AMACR + PSA + / (-)PSA + / (-) 3434ßE12 (-) almost allßE12 (-) almost all p63 (-) almost allp63 (-) almost all Prostein +Prostein + NKX3.1 + NKX3.1 +

Benign Benign ProstateProstate

EpCam (-)EpCam (-) ATM (-) / +ATM (-) / + AMACR (-)AMACR (-) PSA + / (-)PSA + / (-) 3434ßE12 +ßE12 + p63 +p63 + Prostein +Prostein + NKX3.1 + 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

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Urothelial Ca vs Urothelial Ca vs Prostate CaProstate Ca

EMAEMA CK7CK7 P63P63 CK5/6CK5/6 EpCamEpCam CD57CD57 PSAPSA PAPPAP NKX3.NKX3.

11 ProsteiProstei

nn

++ --++ --+/-+/- 00+/-+/- 00+/-+/- 00

-/+-/+ +/-+/- 00 ++

00 +/-+/- 00 ++ 00 ++ 00 ++

Urothelial ca Prostate ca

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

Urothelial ca

Prostate ca

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Ab CocktailsAb Cocktails Breast CaBreast Ca: : ER + GCDFP-15ER + GCDFP-15 MelanomaMelanoma: : MART-1+Melan MART-1+Melan

A+Tyrosine +A+Tyrosine +

PNL2PNL2 Basal Cell MarkerBasal Cell Marker: : p63 + 34betaE12 p63 + 34betaE12

+ +

CK5/6CK5/6 Endocrine Ca:Endocrine Ca: Chrom A+NSE +Chrom A+NSE +

SynaptophysinSynaptophysin Pan SarcomaPan Sarcoma: Vim + Coll IV + CD 99: Vim + Coll IV + CD 99

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

Ad.CaAd.CaECAECA

MUC-1(-)MUC-1(-) ER(-)ER(-) PR(-)PR(-) P16(+)P16(+)

EMMAEMMA MUC-1 (+)MUC-1 (+) ER(+)ER(+) PR(+)PR(+) P16(-)P16(-)

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

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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

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Characteristics of Characteristics of antibodies used for antibodies used for

evaluation.evaluation. Antibody/Clone/Source/Antigen retrieval/DilutionAntibody/Clone/Source/Antigen retrieval/Dilution MUC1 Ma695 Novocastra, Hi temp 1:100MUC1 Ma695 Novocastra, Hi temp 1:100

P16 E6H4 DAKO, TRS 1:25 P16 E6H4 DAKO, TRS 1:25 Vim Vim3B4 DAKO, TRS 1:1000Vim Vim3B4 DAKO, TRS 1:1000

ER 1D5 DAKO, TRS/Vector 1:100ER 1D5 DAKO, TRS/Vector 1:100PR PgR636 DAKO, TRS/Vector 1:200PR PgR636 DAKO, TRS/Vector 1:200

Khoury Khoury et al.et al. BMC Clinical PathologyBMC Clinical Pathology 2006 6:1   doi:10.1186/1472- 2006 6:1   doi:10.1186/1472-6890-6-16890-6-1

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The Cost-Effectiveness of The Cost-Effectiveness of IHCIHC

Why do IHC assay?Why do IHC assay? To increase patient life expectancy by correctTo increase patient life expectancy by correct

diagnosis and treatment plan.diagnosis and treatment plan. To increase diagnostic certaintyTo increase diagnostic certainty To predict patient prognosisTo predict patient prognosis

eg., 35yr old female, history of Br ca, radical eg., 35yr old female, history of Br ca, radical mastectomy performed earlier. Now mastectomy performed earlier. Now diagnosed with cirrhosis and liver mass.diagnosed with cirrhosis and liver mass.

1.1. Meatastatic Ad Ca ?Meatastatic Ad Ca ?2.2. Hepato Cellular Ca ?Hepato Cellular Ca ?

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LastlyLastly

“ “The Diagnostic Power of any The Diagnostic Power of any Immunohistochemical Procedure Immunohistochemical Procedure is no Greater than the wisdom of is no Greater than the wisdom of the Pathologist interpreting it.” the Pathologist interpreting it.”

Dr.Allen GownDr.Allen Gown

Thank You all.Thank You all.