Approach at lymph node pathology and ancillary techniques

Approach at lymph node pathology and ancillary techniques Hans Konrad Müller-Hermelink Institute of Pathology, University of Würzburg Würzburg, Germany

1000 km 400 km

Würzburg

Germany: 80.000.000 population Würzburg: 200.000 population

.contemporary lymphoma diagnosis integrates histology, immunophenotype, (molecular) genetic data as well as clinical features

Frequency of lymph node diseases in primary material Lymphadenitis 6141 56% Metastasis 2658 24% Lymphoma B-cell line: 65% T-cell line: 8% Hodgkin: 27% 1636 15% Granulomatous lymphadenitis 357 3% Other infectious types of lymphadenitis 230 2% Autoimmune disease 28 <1%

Reactive Hyperplasias and Reaction Patterns in different compartments B-cell compartment Follicular hyperplasia Sinusoidal B-cell reaction Marginal zone reaction/ nodules Extrafollicular B-cell activation Plasmacytosis Progressive transformation of germinal centres T-cell compartment Nodular paracortical hyperplasia Diffuse paracortical hyperplasia CD 4 dominant DTH activation CD 8 dominant cytotoxic activation Sinus reactions Sinus histiocytosis Foreign body reaction Mastozytosis Sinus lymphocytosis Macrophage reactions Foreign body reactions Epitheloid cell reactions Purulent granulomas

T-zone CD5

Diffuse paracortical hyperplasia

Diffuse paracortical hyperplasia CD5

T-Zone

Dermatopathic Lymphadenitis CD1a

C CD 8 + cytotoxic hyperplassia in viral lymphadenitis CD 3 CD 8 CD3

CD 8 + cytotoxic hyperplassia in viral lymphadenitis Ki67

Extrafollicular response Follicular response 9113/01 EFH 15310

Naïve B-cell IgM/IgD surface receptor primary follicles mantle zone

Mantle zone: IgD FDC: CD23

Primary immunoresponse antigen proliferating IgM + cells formation of short-lived plasma cells short-lived plasma cell

Primary immunoresponse Brighenti A et al: Histopathology. 2005 Jul;47(1):90-100

Ki-67

CD20 CD3

CD79a IgLC

Secondary immuneresponse antigen Plasma cell protective memory memory cell reactive memory Germinal center reaction Proliferation Affinity maturation by somatic hypermutation Immunglobulin gene class switch >98% of the cells die by apoptosis

Ki67 BCL2

Follicles Medullary cords Extrafoll. activation

r CD3 Donkey anti Rabbit m Ki67 Donkey anti Mouse Donkey anti Goat m CD79a Goat anti FITC

Germinal center reaction Extrafollicular reaction Bild intrafollicular icsat+ cells are CD79a+ IgL+ super 15310_01 Chef spezial Mantel, Keim, Aussen3_Ov4Ausse n CD79a gruen, Igl rot, Icsat blau.jpg CD79a IgL IRF-4

Germinal center reaction Extrafollicular reaction CD79a ICSAT KI67 Ki67 CD79a IRF-4

H1317/01 CD79a IgL CD138 germinal center reaction

Developmental pathways of the B-cell activation IgM + CD27 CD138 IgG + CD27 + CD138 +

antigen antigen plasma cell protective memory memory cell reactive memory short-lived plasma cell

B-cell development in lymph nodes plasma cell protective memory Ki67 CD20 CD79a PAX5 PU1 BCL2 CD10 BCL6 IRF-4 BLIMP-1 IgG CD27 CD138 memory cell reactive memory

Reactive Hyperplasias and Reaction Patterns in different compartments B-cell compartment Follicular hyperplasia Sinusoidal B-cell reaction Marginal zone reaction/ nodules Extrafollicular B-cell activation Plasmacytosis Progressive transformation of germinal centres T-cell compartment Nodular paracortical hyperplasia Diffuse paracortical hyperplasia CD 4 dominant DTH activation CD 8 dominant cytotoxic activation Sinus reactions Sinus histiocytosis Foreign body reaction Mastocytosis Sinus lymphocytosis Macrophage reactions Foreign body reactions Epitheloid cell reactions Purulent granulomas Plasmacytoid monocyte nodules

Sinusoidal B Cell Reaction HE

Sinusoidal B Cell Reaction HE

Sinusoidal B Cell Reaction Giemsa

Sinusoidal B Cell Reaction CD20

Sinusoidal B Cell Reaction CD5

Sinusoidal B Cell Reaction IRF-4 CD27

Sinusoidal B Cell Reaction Ki-67 BCL-2

Differenzierungsantigene Keimzentrumsreaktion Plasmazelle Protektives Gedächtnis Ki67 CD20 CD79a PAX5 PU1 BCL2 CD10 BCL6 IRF-4 BLIMP-1 IgG CD27 CD138 Memoryzelle Reaktives Gedächtnis

Monocytoid (Sinusoidal) B Cell Reaction Acute prefollicular B cell hyperplasia filling marginal and intermediate sinuses ( different from marginal zone B cell hyperplasia) Acute and subacute Infections: e.g. Toxoplasma gondii HIV EBV CMV Very unusual in malignant lymphoma with the exeption of very rare cases of monocytoid B Cell lymphoma

Piringer Lymphadenitis

H1645/01 Giemsa x20

Reactive Hyperplasias and Reaction Patterns in different compartments B-cell compartment Follicular hyperplasia Sinusoidal B-cell reaction Marginal zone reaction/ nodules Extrafollicular B-cell activation Plasmacytosis Progressive transformation of germinal centres T-cell compartment Nodular paracortical hyperplasia Diffuse paracortical hyperplasia CD 4 dominant DTH activation CD 8 dominant cytotoxic activation Sinus reactions ( medullary) Sinus histiocytosis Foreign body reaction Mastocytosis Sinus lymphocytosis Macrophage reactions Foreign body reactions Epitheloid cell reactions Purulent granulomas Plasmacytoid monocyte nodules

Plasmacytoid monocytes Cytokine producing monocyte/dendritic cell population ( IFN-a) in response to viral challenge Characteristic phenotype (CD68+, CD4+, CD56+/-, CD123+, BDCA2+) Seen in 16% of non-specific lymphadenitis cases, particularly prominent in Kikuchi lymphadenitis Related to CD 4+,CD56+ hematodermic neoplasia)

H1170/09 HE x40

H1170/09 Giemsa x40

H1170/09 CD68 x40

H8847/00 HE x40

H8847/00 CD68 x40

Reactive Hyperplasias and Reaction Patterns in different compartments B-cell compartment Follicular hyperplasia Sinusoidal B-cell reaction Marginal zone reaction/ nodules Extrafollicular B-cell activation Plasmacytosis Progressive transformation of germinal centres T-cell compartment Nodular paracortical hyperplasia Diffuse paracortical hyperplasia CD 4 dominant DTH activation CD 8 dominant cytotoxic activation Sinus reactions Sinus histiocytosis Foreign body reaction Mastozytosis Sinus lymphocytosis Macrophage reactions Foreign body reactions Epitheloid cell reactions Purulent granulomas

Human Marginal Zone B Cells IgM+,IgD+,CD27+,(CD21+, CD1c +) Prediversification ( hypermutation of BCR) occuring at early age, polyclonal, no selection on antigen priming, no memory (?) Reaction to TI antigen stimulation, but also to TD antigens ( in humanized SCID mice) Non-cognate interaction with Th cells may induce IgH switch Don t enter follicular reactions No AID ( human splenic marginal zone B cells) Unclear whether seperate naive B cell subset or specialized antigen expanded memory population ( e.g. in GALT) K.Willenbrock et al. Eur. J. Immunol. (2005) 35:3002-7 St.G.Tangye, K.L.Good : J.Immunol (2007) 179: 13-19 L.Moens, et al. :J.Immunol. (2008)181: 5306-12;D.Tarlington JEM (2008)205: 1251-54 S.Pillai, A. Cariappa:Nature Reviews Immunol (2009) 9: 767-777 J.-C.Weill,S.Weller, C.-A.Reynaud : Annu.Rev.Immunol. (2009) 27:267-85

The human marginal zone contains heterogeneous B cell subpopulations; but man are not mice Fo B memory cell reactive memory plasma cell protective memory? Mz B? short-lived plasma cell

Marginal zone Reaction( hyperplasia) Marginal zone B cells are heterogeneous Marginal zone has been defined in the spleen, where the outer extrafollicular response is a prefollicular reaction mostly to TI antigens. Similar reactions and structural findings do exist in lymph nodes Better understanding necessary to define the normal counterparts of nodal and splenic MZBL

C3d receptor (CD21)

marginal zone

CD27 expression in B-Lymphocytes Marker for postfollicular cells Somatic hypermutations only in CD27 + B-cells Immunoglobulin isotype switch in CD27+ cells Immunoglobulin production more efficient Cord blood does not contain CD27+ B-cells Agematsu K: Eur J Immunol 1997;27:2075 Klein U: J Exp Med 1998;188:1679 Weller S: PNAS 2001;98: 1166 Nagumo H: Blood 2002;99:567

Successive stem cells and differentiation phases in normal B cell development Bone marrow Lymphoid tissue Bone marrow T, NK CD5 +? pluripotent hämopoetic lymphoid VDJ-rearrangement Stem cell Stem cell mature B cell memory B cell plasma cell germinal center extrafollicular activation 1 Heterogeneity 2 Specificity 3 Effectors 4 Defense Organ

Successive stem cells and differentiation phases in normal B cell development Bone marrow Lymphoid tissue Bone marrow T, NK CD5 +? Marginal zone B cell pluripotent hämopoetic lymphoid VDJ-rearrangement Stem cell Stem cell mature B cell memory B cell plasma cell germinal center extrafollicular activation 1 Heterogeneity 2 Specificity 3 Effectors 4 Defense Organ

Antigen Antigen Plasma Cell protective memory Memory B cell reactive memory B CLL B CLL Akute Lymphoid Leukemia Mantle Cell- Lymphoma Follicular Lymphoma Marginal Zone- B cell Lymphoma Plasmocytoma MM Diffuse Large B Cell Lymphoma Germinal Center B Cell Type Activated B Cell Type

Immunophenotypic analysis Detection of a clonal B- or T- cell population Detection of genetic alteration

Frequently used immunophenotypic markers Lineage specific Non lineage specific Tumor marker? B-cell T-cells Aberrant subcellular and tissue distribution? Abnormal constellations? PAX5 CD20 CD19 CD23 CD79a CD10 BCL6 MUM1 CD138 κ/λ IgM IgG IgD IgA CD3 CD2 CD5 CD7 CD4 CD8 TdT PD1 Perforin bf1 TdT CD30 Ki67 ALK1 CD56 CD15 BCL2 CyclinD1 EMA

Immunophenotypic analysis Flow cytometric analysis of the cell suspension Immunohistochemistry of the tissue section Peripheral blood, bone marrow, body fluids Flexibility Accurate quantitative analysis Simultaneous detection of several markers Topographic distribution of immunostained cell population Routine paraffin section Specimens with a small percentage of tumor cells (HD)

Chronic lymphocytic leukaemia CD20 Ki-67 CD5 CD23

Mantle cell lymphoma H&E Cyclin D1 CD5 CD23

Follicular lymphoma Ki67 BCL2

CD20 AE1/AE3

Anaplastic large cell lymphoma CD30 ALK1

NLPHL Classical HL CD30

NLPHL Classical HL CD20

A minimum of Immunehistochemistry for lymphoma classification?

CD20 / CD5 Distribution and pattern of B and T cell areas Basic structure of the lymph node IgD Follicle Parafollicular pulp Coexpression of CD5 in CLL and Mantle cell lymphoma Mantle zone Expression in Mantle cell lymphoma CD23 Follicular dendritic cells Marginal zone (inconsistent) Coexpression of CD23 in CLL

Ki-67 where the music plays Abnormal activities BCL-2 High proliferation in extrafollicular areas Low proliferation within the follicles Expression in all B cells outside of germinal centers Follicular expression in follikular lymphoma Does not distinguisish different types of indolent B cell lymphoma

CD30 Hodgkin lymphoma Anaplastic large cell lymphoma Weak expression also in plasma cells (intern control) CD15 Expression in granulocytes Hodgkin lymphoma

Immunehistochemistry reactive l.n. CD5 CD20 CD23 Ki-67 CD30 confirmation T cells B cells FDC(marginal zone) Follicle Variabel CLL + + + 10-20% MCL + + (FDC remnants) B blasts Richter Syndrom e -40% - FL - + Follicles - - DLBCL - + - (+) (Cyclin D1, p27, IgD) bcl-2 (CD10/ bcl-6) chl rosettes -/+ - Variabel + CD15 PTCL +/- - FDC (AILT) Variabel -/+?

Cave! IHC confirms or rejects diagnoses favoured by morphology CD20 CD5 + CD23 - + + - - IgD + Ki67 <20% Ki67 variabel Cyclin D1+ B-CLL MCL Ki67 Ki67 >99% Bcl2 CD10+ Burkitt CD30 - + ALCL Hodgkin DLBCL CD23 FDC- FDC Ki67 40-80% BCL2+ CD10+ Ki67 variabel DLBCL FL T cell lymphoma Recurrance after RituxiMab Extranodal Exclude follicular colonisation MZL

Immunehistochemistry CD79a Recurrences after Rituximab treatment Later differentiation antigen of B cells CD3 Comparison with CD5 e.g coexpression im B cells IgD L.n. architecture, mantle zone, NLPHL k/l Clonality, rarely characterizes lymphoma type Cyclin D1 Note intern control= endothelia CD10/ BCL-6 follicles BCL-2 Expression in all indolent B cell lymphoma Over expression in follicles of FL LMP-1 EBV in mononucleosis, Hodgkin lymphoma, PTLD etc. CD138 Plasma cells

Molecular studies Important help in the diagnosis of malignant lymphoma Morphological approach essential : no tumor diagnosis on molecular findings alone!!!! Diagnostic approach is correlated to treatment options ( e.g. CD20 and RituxiMab treatment)

Molecular studies B-cell clonality T-cell clonality IgH PCR Microdissection and IgH PCR Quantitative RT-PCR for kappa and lambda light chains Southern blot TCR PCR a.o. Southern blot Cyclin D1 overexpression Quantitative PCR Mutation analysis Infectious agents Chromosomal Translocations sequencing ( e.g. c-kit, p53, IgH) PCR ( e.g.mycobacteria, Chlamydia) Southern blot, PCR, RT-PCR, FISH

The Immunglobulin Receptor a fingerprint of individual clonal development and current activities of B-lymphocytes Clonality and clonal relationship Somatic hypermutation Ongoing mutations VH-family usage Antigen selection Minimal residual disease Idiotypic therapeutic targets

Analysis of Somatic Hypermutation of IgVH Genes in B-Cell Lymphoma Lymphoma Somatic Mutation Ongoing Mutations B-CLL + (50%) - B-PLL +??? MCL -/+ - FL +++ + MZBL, MALT ++ + MZBL, splenic ++/- - MZBL, nodal ++/- - HCL ++ - Plasmacytoma ++ -

Recurrent chromosomal translocations are very frequent in mature B-cell lymphoma Chromosomal translocation Gene function B-NHL subcategory frequency t(14;18)(q32;q21) and variants BCL2 anti-apoptotic protein FL DLBCL CLL 80-90% 20-30% <1% t(1;22)(q22;q11) FCGR2B low affinity Ig Fc receptor FL, DLBCL <1% t(8;14)(q24;q32) and variants MYC transcription factor BL DLBCL FL MM ~100% 5-10% <1% t(3;14)(q27;q32) and variants BCL6 transcriptional repressor DLBCL FL 40%?% t(11;14)(q13;q32) CCND1 G1 cyclin MCL MM >95% ~20% t(9;14)(p13;q32) PAX5 transcription factor variant SLL not established t(11;18)(q21;q21) API2/ MALT1 anti-apoptosis protein MALT ~30%* t(14;15)(q32;q11-13) BCL8 DLBCL ~4% t(1;14)(q21;q32) MUC1 cell surface receptors DLBCL <1% t(1;14)(p22;q32) BCL10 apoptosis regulatory protein MALT t(14;18)(q32;q21) MALT1 MALT ~20%* t(14;19)(q32;q13) BCL3 IκB C L L / S L L < 5 % t ( 4 ; 1 4 ) ( p 1 6 ; q 3 2 ) F G F R 3 / M M S E T M M 1 5-2 0 % t ( 1 4 ; 1 6 ) ( q 3 2 ; q 2 3 ) c - m a f t r a n s c r i p t i o n f a c t o r M M 2-1 0 % a n d v a r i a n t t ( 6 ; 1 4 ) ( p 2 1 ; q 3 2 ) C y c l i n D 3 C e l l c y c l e p r o t e i n M M 3-4 % t ( 6 ; 1 4 ) ( p 2 5 ; q 3 2 ) M U M 1 / I R F - 4 i n t e r f e r o n r e g u l a t o r y f a c t o r M M n o t e s t a b l i s h e d d e r 7 q 2 1 C D K 6 c e l l c y c l e k i n a s e s p l e n i c M Z L 6 0-7 0 %

t(14;18) t(11;14) t(8:14)/ebl t(3;14)? t(8:14)/sbl t(4;14) t(11;14) Somatic Hypermutation Germinal Center Class Switching V(D)J recombination FDC T Plasma cell Naive B cell apoptosis apoptosis Memory B cell ALL MCL B-CLL(subtype) BL FL DLBCL (subset) MZL B-CLL (subset) DLBCL (subset) MM HCL? secondary genetic alterations: genomic gains,amplifications, deletions, mutations

Mantle cell lymphoma Cyclin D1 deregulation

Interphase Fluorescence In Situ Hybridization

Fluorescence in situ hybridization Relatively simple technique for targeted detection of genetic aberrations Non dividing or terminally differentiated cells, non vital cells, when dividing cells do not represent the malignant clone Provides information on single cell level

Commercially available probes

FISH on routinely processed paraffin tissue samples not restricted on the availability of fresh or frozen material

FISH on routinely processed paraffin tissue samples Nuclei preparation Paraffin sections Avoid cutting and overlapping artifacts easy for evaluation numerical aberrations, deletions, amplification complex patterns detection of the breakpoint in the histological context needs less patients material control on the presence of the tumor population in the sample

FISH patterns on routine paraffin tissue sections Intact nuclei 3-4µm Tissue section A B C B D E F G H Normal tissue Tumor tissue

FISH on paraffin sections - signal patterns

Cut off values for different Vysis probes on routine paraffin section Reactive samples Bcl2/IgH (%cells with aberrant pattern) MYC BAP (%cells with aberrant pattern) MALT1 BAP (%cells with aberrant pattern) IGH BAP (%cells with aberrant pattern) mean 11 0,5 4 9,4 SD 2 0,95 1,58 3,4 Cut off 17 4 0 9 20 Bcl1/IgH (%cells with aberrant pattern)

FICTION combined ICH and FISH

CyclinD1

kappa

FICTION Kappa/CyclinD1 BAP Kappa/CyclinD1 Double immunofluorescence

How to integrate the FISH data into histopathology diagnosis? Genomic abnormality = malignancy Genomic abnormality = lymphoma type FL t(14;18)(q32;q21) + (85%) bcl2 protein + FL t(14;18)(q32;q21) bcl2 protein +/- DLBCL t(14;18)(q32;q21) + GCB expression profile Bcl2 +/- favorable prognosis t(14;18)(q32;q21) bcl2 protein + ABC expression profile poor prognosis

Interphase FISH analysis is preferable method for detection of the chromosomal translocations as many breakpoints are dispersed over the large genomic area Both practical and biological considerations are in favor of FISH segregation assay (BAP) in which two differently labeled probes segregate upon a break Introduction of reliable hybridization protocols for paraffin tissue allows the usage of FISH in routine pathology settings Things should be made as simple as possible, but not any simpler. Albert Einstein