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How Many Markers is How Many Markers is Enough? Enough?

HematopathologyHematopathology

Emina Emilia Torlakovic, MD, PhDEmina Emilia Torlakovic, MD, PhDCollege of MedicineCollege of Medicine

University of SaskatchewanUniversity of SaskatchewanSaskatoon, SK, CanadaSaskatoon, SK, Canada

Kremer M, Quintanilla-Martinez L, Nahrig J, von Schilling C, Fend F.Immunohistochemistry in bone marrow pathology: a useful adjunct for morphologic diagnosis. Virchows Arch. 2005 Oct 18;:1-18

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IHC in BM pathologyIHC in BM pathology

Acute leukemia (ALL and AML)Acute leukemia (ALL and AML) Blasts estimates/countsBlasts estimates/counts

–– MDS, MDS/MPN, MPNMDS, MDS/MPN, MPN Malignant lymphomaMalignant lymphoma Metastatic tumorsMetastatic tumors Systemic mastocytosisSystemic mastocytosis

OtherOther

Acute LeukemiaAcute Leukemia

CD34, TdT, MPO, CD33, CD117, F8CD34, TdT, MPO, CD33, CD117, F8--ra, ra, CD61 (or CD42b), HgbCD61 (or CD42b), Hgb--A, A, GlyGly--C, PaxC, Pax--5, CD10, CD79a, CD20, CD45, CD35, CD10, CD79a, CD20, CD45, CD3

To consider:To consider: NPM1, CD68, CD63, NPM1, CD68, CD63, lysozyme, CD15, CD123, CD99, CD31lysozyme, CD15, CD123, CD99, CD31

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MDS and MDS/MPNMDS and MDS/MPN

CD34, CD117, CD61 (or CD42b), MCTCD34, CD117, CD61 (or CD42b), MCT

To consider:To consider: MPO, CD33, MCT, HgbMPO, CD33, MCT, Hgb--A, A, Parvovirus B19Parvovirus B19

Myeloproliferative Neoplasms Myeloproliferative Neoplasms (MPN)(MPN)

CD34, MPO, CD61 (or CD42b or F8CD34, MPO, CD61 (or CD42b or F8--ra), PGra), PG--M1, HgbM1, Hgb--AA

To consider:To consider: CD33, TdT, CD3, CD20, CD33, TdT, CD3, CD20, PaxPax--55

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CD34 Counts & EstimatesCD34 Counts & Estimates

(%) in 500 (%) in 500 cell countcell count

> 3 pos. cells> 3 pos. cells > 5 pos. cells> 5 pos. cells

Mature BMature B--cell Neoplasmscell Neoplasms

CD3, CD20, CD10, CD5, cyclin D1, CD3, CD20, CD10, CD5, cyclin D1, CD23, BclCD23, Bcl--66

To consider:To consider: CD79a, IgM, IgD, CD25, CD79a, IgM, IgD, CD25, AnnexinAnnexin--1, Bcl1, Bcl--2, k, l, CD138, CD72, 2, k, l, CD138, CD72, BclBcl--3, ZAP70, CD433, ZAP70, CD43

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CD20 and CD3 are always CD20 and CD3 are always interpreted togetherinterpreted together

CD20 CD20 ≠≠ PaxPax--5 5 ≠≠ 79a79a

CD20 CD20 ≠≠ PaxPax--55

J Pathol

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Pax-5 in FL

CD20: LymphomaCD20: Lymphoma

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MCL HCL MM

Cyclin D1Cyclin D1

Mature TMature T--cell Neoplasmscell Neoplasms

CD2, CD3, CD4, CD5, CD7, CD8, CD2, CD3, CD4, CD5, CD7, CD8, CD56, CD57, TIACD56, CD57, TIA--1, 1, grenzymegrenzyme B, EBVB, EBV

To consider:To consider: MUM1, CD45RO, BclMUM1, CD45RO, Bcl--6, 6, CD30, ALKCD30, ALK--1, perforin1, perforin

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Hodgkin LymphomaHodgkin Lymphoma

NLPHL:NLPHL: PaxPax--5, CD20, CD3, Bcl5, CD20, CD3, Bcl--6, Oct6, Oct--2, CD572, CD57

Classical:Classical: For presentation in the BM: CD30, CD15, CD3, For presentation in the BM: CD30, CD15, CD3,

CD20, PaxCD20, Pax--5, MUM15, MUM1–– To considerTo consider: EBV, PU.1, Oct2, BOB.1: EBV, PU.1, Oct2, BOB.1

For staging when malignant cells present: CD30, For staging when malignant cells present: CD30, CD15CD15

For staging when abnormal cells not For staging when abnormal cells not morphologically obvious: CD3, Paxmorphologically obvious: CD3, Pax--5, CD305, CD30

Plasma Cell DisordersPlasma Cell Disorders

CD138, k, l, CD45, CD56, CD20CD138, k, l, CD45, CD56, CD20

To consider:To consider: KiKi--67, EBV, IgM, IgD, 67, EBV, IgM, IgD, IgA, IgG, IgE, PaxIgA, IgG, IgE, Pax--5, CD117, cyclin 5, CD117, cyclin D1, HHVD1, HHV--8, EBER8, EBER

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Histiocytic and Dendritic Cell Histiocytic and Dendritic Cell NeoplasmsNeoplasms

CD68, CD163, PU.1, CD45, CD21, CD68, CD163, PU.1, CD45, CD21, CD23, CD35, CD123, SCD23, CD35, CD123, S--100, CD1a, 100, CD1a, vimentinvimentin

To consider:To consider: fascinfascin, , langerinlangerin, , lysozyme, TCL1lysozyme, TCL1

MastocytosisMastocytosis

MCT, CD25, CD117MCT, CD25, CD117

To consider: CD34, CD3, CD20To consider: CD34, CD3, CD20

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LymphomaLymphoma

WHO 2008 WHO 2008 ClassificationClassification

–– Selection of Selection of markers markers depends on criteria depends on criteria required required for for the classificationthe classification

–– Evidence based medicineEvidence based medicine

Basic PanelBasic Panel

CD3, CD20, BclCD3, CD20, Bcl--2, CD138, kappa, 2, CD138, kappa, lambdalambda

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Lymphoplasmacytic

lymphoma

Always Interpreted Always Interpreted TogetherTogether

CD45RO-UCHL1≠CD3

CD3

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Cytoplasmic CD3

•T-lymphoblastic•T/NK lymphomas

T/HRBCL

CD3

CD20

CD8

CD8

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CD79aCD75

CD21CD20

CD20

CD75

CD79a

CD75

NLPHL

CD20

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CD57

Oct-2

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KiKi--67 in 67 in HematopathologyHematopathology Burkitt lymphoma: Burkitt lymphoma: ““nearly 100%nearly 100%”” ((class Iclass I)) Mantle cell lymphoma: > 40% adverse prognostic Mantle cell lymphoma: > 40% adverse prognostic

indicator (indicator (class IIclass II)) ALL (low ALL (low –– better prognosis): higher in adults than better prognosis): higher in adults than

in children and higher in Tin children and higher in T--ALL than in BALL than in B--ALL; good ALL; good prognosis ALL <25% (prognosis ALL <25% (class IIclass II))

Very high in ALPS (Very high in ALPS (class Iclass I)) Low in most lowLow in most low--grade LPD (grade LPD (class Iclass I)) Plasmablastic lymphoma: minimum 75%, usually Plasmablastic lymphoma: minimum 75%, usually

>90% (>90% (class Iclass I)) OtherOther……

Cyclin D1 in Mantle Cell Lymphoma Cyclin D1 in Mantle Cell Lymphoma

Control

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ALK-1 in ALCL with t(2;5)

WHAT IS YOUR DIAGNOSIS?

Skin Bx:Blastic morphology, TdT+, Pax-5+,CD45-

Answer:Merkel Cell Carcinoma (CK20+)

Pax-5

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Critical BCritical B--Cell MarkersCell Markers

–– CD5CD5–– CD10CD10–– CD20CD20–– CD21CD21–– CD23CD23–– CD43CD43–– CD45CD45–– CD79aCD79a

–– PaxPax--55–– CD138 & CD138 & –– BclBcl--22–– BclBcl--66–– Cyclin D1Cyclin D1–– KiKi--6767–– TdTTdT–– CD34CD34

–– EBV (EBER or LMP1)EBV (EBER or LMP1)

Additional Important BAdditional Important B--Cell MarkersCell Markers

HHVHHV--8 (PEL, CD)8 (PEL, CD) CD30CD30 IgMIgM IgG, IgD, IgAIgG, IgD, IgA MUM1MUM1 PU.1PU.1

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Critical TCritical T--Cell MarkersCell Markers

–– CD56CD56–– ALKALK–– TdT TdT –– CD34CD34–– CD4CD4–– CD8CD8–– TIATIA--11–– Granzyme B/perforinGranzyme B/perforin

–– CD2, CD3, CD5, CD7CD2, CD3, CD5, CD7–– CD10, CD57, PDCD10, CD57, PD--1, CLCX1, CLCX--11–– CD21CD21–– CD30CD30–– CD43CD43–– CD45, CD45ROCD45, CD45RO

–– EBV (EBER or LMP1)EBV (EBER or LMP1)

Hodgkin LymphomaHodgkin Lymphoma

–– CD3 and CD20CD3 and CD20–– CD15 and CD30CD15 and CD30–– PaxPax--5, MUM1, and Oct5, MUM1, and Oct--22–– CD21 and CD23CD21 and CD23–– CD45CD45–– BclBcl--6, EMA, CD576, EMA, CD57

–– EBV (EBER or LMP1)EBV (EBER or LMP1)

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CD20 (Membranous)CD20 (Membranous)

Most commonly used panMost commonly used pan--B antibodyB antibody

Some BSome B--cell lymphomas are defined cell lymphomas are defined by the lack of expression of CD20 by the lack of expression of CD20 (PLASMABLASTIC LYMPHOMA)(PLASMABLASTIC LYMPHOMA)

Some myelomas are defined by Some myelomas are defined by expression of CD20 (CD20+ MM)expression of CD20 (CD20+ MM)

It is not necessarily negative in It is not necessarily negative in cHLcHL Special attention to Rituximab TXSpecial attention to Rituximab TX

CD79a (Cytoplasmic)CD79a (Cytoplasmic)

It is considered as a backIt is considered as a back--up B cell antibody; up B cell antibody; It is very valuable in providing additional It is very valuable in providing additional informationinformation

Expressed at all stages of BExpressed at all stages of B--cell differentiation cell differentiation (best marker for lymphoplasmacytic (best marker for lymphoplasmacytic lymphoma)lymphoma)

It is not lineage marker; Expressed in some It is not lineage marker; Expressed in some AML, and also some TAML, and also some T--ALLALL

Malignant plasma cells express CD79a variablyMalignant plasma cells express CD79a variably Very unusual to see in Very unusual to see in cHLcHL; CD20 is much ; CD20 is much

more common than CD79amore common than CD79a

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PaxPax--5 and MUM1/IRF4 5 and MUM1/IRF4 (Nuclear)(Nuclear)

Not really backNot really back--up markers up markers PaxPax--5 widely expressed 5 widely expressed MUM1 very selectively expressed MUM1 very selectively expressed

(post(post--GC marker)GC marker) Very useful for HL, ALL, CD20Very useful for HL, ALL, CD20--

negative Bnegative B--cell lymphomas cell lymphomas

Kappa and LambdaKappa and Lambda

Demonstrate cytoplasmic light chains in Demonstrate cytoplasmic light chains in plasma cells and some Bplasma cells and some B--cell lymphomascell lymphomas–– MM, LPL, IBL, MZLMM, LPL, IBL, MZL

PitfallsPitfalls–– Extremely difficult to optimize to detect surface Extremely difficult to optimize to detect surface

light chain and to reliably interpret results of light chain and to reliably interpret results of cytoplasmic light chainscytoplasmic light chains

–– Consider in situ hybridization for light chain Consider in situ hybridization for light chain mRNA: the sensitivity is the same but the mRNA: the sensitivity is the same but the specificity is higherspecificity is higher

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CD21, CD23 (Pan B) CD21, CD23 (Pan B)

Mainly for detection of follicular dendritic cells Mainly for detection of follicular dendritic cells ((FDCsFDCs))–– Follicular lymphoma (! needle core bx)Follicular lymphoma (! needle core bx)–– AITCL (relationship to vascular structures)AITCL (relationship to vascular structures)–– FLg3 vs. DLBCLFLg3 vs. DLBCL–– cHLcHL–– Follicular TFollicular T--cell lymphomacell lymphoma–– Dendritic cell tumorsDendritic cell tumors

Chronic lymphocytic leukemiaChronic lymphocytic leukemia Rare in DLBCLRare in DLBCL

BclBcl--2 (Cytoplasmic)2 (Cytoplasmic)

CRITICAL MARKER FOR:CRITICAL MARKER FOR:–– FL vs. reactive follicular hyperplasiaFL vs. reactive follicular hyperplasia

BurittBuritt lymphoma vs, DLBCL (along with Kilymphoma vs, DLBCL (along with Ki--67)67)–– BL is BclBL is Bcl--2 NEGATIVE!2 NEGATIVE!

PitfallsPitfalls–– Negative in minority of Negative in minority of FLsFLs (just like CD10)(just like CD10)–– Negative in primary cutaneous FLNegative in primary cutaneous FL–– Colonizing marginal zone cells will be bclColonizing marginal zone cells will be bcl--2(+): 2(+):

correlate with Bclcorrelate with Bcl--6 and CD10 expression.6 and CD10 expression.

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CD5 (Membranous)CD5 (Membranous)

Essential in the panel for evaluation Essential in the panel for evaluation of small Bof small B--cell cell lymphoprolifertivelymphoprolifertivedisorders (CLL & MCL vs. MZL, LPL, disorders (CLL & MCL vs. MZL, LPL, FL)FL)

CLL > MCLCLL > MCL CD5+ DLBCL is very aggressive CD5+ DLBCL is very aggressive

diseasedisease Loss of CD5 in TLoss of CD5 in T--cell lymphomascell lymphomas

CD4 and CD8 CD4 and CD8 (Membranous)(Membranous) Double positive in TDouble positive in T--ALLALL Often mixed even in TOften mixed even in T--cell lymphomascell lymphomas Reactive CD8 cells can predominate Reactive CD8 cells can predominate

and can have very atypical and can have very atypical morphology (T/HRBCL)morphology (T/HRBCL)

CD4 prim Ab may be difficult to CD4 prim Ab may be difficult to optimize in FF/PE tissueoptimize in FF/PE tissue

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CD56CD56

All nasalAll nasal--type NK/T cell lymphomastype NK/T cell lymphomas Positive in aggressive NKPositive in aggressive NK--cell leukemiacell leukemia Positive in the rare blastic plasmacytoid Positive in the rare blastic plasmacytoid

dendritic cell neoplasm (CD4+, CD56+ dendritic cell neoplasm (CD4+, CD56+ hematodermichematodermic neoplasm)neoplasm)

Positive in MMPositive in MM Rarely positive in TRarely positive in T--LGLLGL

CD30 CD30 (Golgi, (Golgi, CyotplasmicCyotplasmic, Membranous), Membranous)

Typically used for ALCL, HL, CD20Typically used for ALCL, HL, CD20--DLBCL,DLBCL, Also expressed on reactive immunoblasts both in Also expressed on reactive immunoblasts both in

T and B immunoblastsT and B immunoblasts DoesnDoesn’’t work well in B5 fixed tissues; t work well in B5 fixed tissues; NOTE: NOTE:

CD15CD15 doesndoesn’’t work so well in formalint work so well in formalin--fixed fixed tissuestissues

Embryonal CaEmbryonal Ca Many large cell lymphomas in subpopulation of Many large cell lymphomas in subpopulation of

cellscells Often found together with MUM1 (ALCL, Often found together with MUM1 (ALCL, cHLcHL))