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 1636 15%
B-cell line: 65%
T-cell line: 8%
Hodgkin: 27%

Granulomatous lymphadenitis 357 3%

Other infectious types of
lymphadenitis 230 2%
Autoimmune disease 28 <1%
 Reactive Hyperplasias and Reaction
Patterns in different compartments
Sinus reactions
B-cell compartment
• Sinus histiocytosis
• Follicular hyperplasia
• Foreign body reaction
• Sinusoidal B-cell reaction
• Mastozytosis
• Marginal zone reaction/ nodules
• Sinus lymphocytosis
• Extrafollicular B-cell activation
Macrophage reactions
• Plasmacytosis
• Foreign body reactions
• Progressive transformation of
germinal centres • Epitheloid cell reactions
T-cell compartment • Purulent granulomas
• Nodular paracortical hyperplasia
• Diffuse paracortical hyperplasia
– CD 4 dominant DTH activation
– CD 8 dominant cytotoxic
activation
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

• EFH 15310
9113/01
Naïve B-cell
IgM/IgD surface receptor
primary follicles
mantle zone
Mantle zone: IgD FDC: CD23
 Primary immunoresponse
proliferating IgM+ cells
antigen 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
 Donkey
r CD3 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
plasma cell
protective memory
antigen

memory cell
reactive memory

short-lived plasma cell

 B-cell development in lymph nodes
plasma cell
protective memory

memory cell
reactive memory
Ki67
CD20
CD79a
PAX5
PU1
BCL2
CD10
BCL6
IRF-4
BLIMP-1
IgG
CD27
CD138
 Reactive Hyperplasias and Reaction
Patterns in different compartments
Sinus reactions
B-cell compartment
• Sinus histiocytosis
• Follicular hyperplasia • Foreign body reaction
• Sinusoidal B-cell reaction • Mastocytosis
• Marginal zone reaction/ nodules • Sinus lymphocytosis
• Extrafollicular B-cell activation Macrophage reactions
• Plasmacytosis • Foreign body reactions
• Progressive transformation of • Epitheloid cell reactions
germinal centres • Purulent granulomas
T-cell compartment • Plasmacytoid monocyte nodules
• Nodular paracortical hyperplasia
• Diffuse paracortical hyperplasia
– CD 4 dominant DTH activation
– CD 8 dominant cytotoxic activation
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

Memoryzelle
Reaktives Gedächtnis
Ki67
CD20
CD79a
PAX5
PU1
BCL2
CD10
BCL6
IRF-4
BLIMP-1
IgG
CD27
CD138
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
Sinus reactions ( medullary)
B-cell compartment
• Sinus histiocytosis
• Follicular hyperplasia • Foreign body reaction
• Sinusoidal B-cell reaction • Mastocytosis
• Marginal zone reaction/ nodules • Sinus lymphocytosis
• Extrafollicular B-cell activation Macrophage reactions
• Plasmacytosis • Foreign body reactions
• Progressive transformation of • Epitheloid cell reactions
germinal centres • Purulent granulomas
T-cell compartment • Plasmacytoid monocyte nodules
• Nodular paracortical hyperplasia
• Diffuse paracortical hyperplasia
– CD 4 dominant DTH activation
– CD 8 dominant cytotoxic
activation
 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
Sinus reactions
B-cell compartment
• Sinus histiocytosis
• Follicular hyperplasia
• Foreign body reaction
• Sinusoidal B-cell reaction
• Mastozytosis
• Marginal zone reaction/ nodules
• Sinus lymphocytosis
• Extrafollicular B-cell activation
Macrophage reactions
• Plasmacytosis
• Foreign body reactions
• Progressive transformation of
germinal centres • Epitheloid cell reactions
T-cell compartment • Purulent granulomas
• Nodular paracortical hyperplasia
• Diffuse paracortical hyperplasia
– CD 4 dominant DTH activation
– CD 8 dominant cytotoxic
activation
 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

memory cell
Fo B 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 lymphoid
hämopoetic VDJ-rearrangement

Stem cell Stem cell mature memory plasma cell

B cell B cell
extrafollicular
germinal center
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

Marginal zone B cell

T, NK CD5+ ?

pluripotent lymphoid
hämopoetic VDJ-rearrangement

Stem cell Stem cell mature memory plasma cell

B cell B cell
extrafollicular
germinal center
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 Mantle Cell- Follicular Marginal Zone- Plasmocytoma

Lymphoid Lymphoma Lymphoma B cell Lymphoma MM
Leukemia

Diffuse Large B Cell Lymphoma

Germinal Center Activated B

B Cell Type 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 PAX5 CD3 TdT

and tissue distribution? CD20 CD2 CD30
CD19 CD5 Ki67
•Abnormal CD23 CD7 ALK1
constellations? CD79a CD4 CD56
CD10 CD8 CD15
BCL6 TdT BCL2
MUM1 PD1 CyclinD1
CD138 Perforin EMA
κ/λ bF1
IgM
IgG
IgD
IgA
 Immunophenotypic analysis

Flow cytometric analysis of the Immunohistochemistry of the

cell suspension tissue section

Peripheral blood, bone marrow, Topographic distribution of

body fluids immunostained cell population

Flexibility Routine paraffin section

Accurate quantitative analysis Specimens with a small percentage

of tumor cells (HD)
Simultaneous detection of several
markers
 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
• Follicle
• Parafollicular pulp
– Coexpression of CD5 in CLL and Mantle cell lymphoma
• IgD
– 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
• High proliferation in extrafollicular areas
• Low proliferation within the follicles

• BCL-2
– 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
CD5 CD20 CD23 Ki-67 CD30 confirmation
reactive FDC(marginal Follicle
T cells B cells B blasts
l.n. zone) Variabel
Richter
CLL + + + 10-20% Syndrom
e
(FDC (Cyclin D1,
MCL + + remnants)
-40% - p27, IgD)
bcl-2
FL - + Follicles  -  - (CD10/ bcl-6)

DLBCL - + -  (+)
cHL rosettes -/+ - Variabel + CD15

PTCL +/- - FDC (AILT) Variabel -/+ ?

 Cave!
IHC confirms or rejects diagnoses favoured by
morphology
Ki67 <20%
CD20 + CD5 + CD23 + B-CLL
- - -
IgD + Ki67 variabel
MCL
Cyclin D1+

 Ki67 >99%

Ki67 Bcl2 – CD10+ Burkitt


 Ki67 40-80%
CD30 + ALCL DLBCL
- Hodgkin
DLBCL CD23 FDC BCL2+ CD10+ FL
Ki67 variabel
FDC-

Extranodal
T cell lymphoma MZL
Exclude follicular
Recurrance after RituxiMab colonisation
• CD79a
Immunehistochemistry
– 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 IgH PCR
Microdissection and IgH PCR
Quantitative RT-PCR for kappa and lambda
light chains
Southern blot

T-cell clonality TCR  PCR a.o.

Southern blot

Cyclin D1 overexpression Quantitative PCR

Mutation analysis sequencing ( e.g. c-kit, p53, IgH)

Infectious agents PCR ( e.g.Mycobacteria, Chlamydia)

Chromosomal
Translocations 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 ”Ongoing”

Mutation 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) BCL2 anti-apoptotic protein FL 80-90%

and variants DLBCL 20-30%
CLL <1%

t(1;22)(q22;q11) FCGR2B low affinity Ig Fc receptor FL, DLBCL <1%

t(8;14)(q24;q32) MYC transcription factor BL ~100%

and variants DLBCL 5-10%
FL <1%
MM

t(3;14)(q27;q32) BCL6 transcriptional repressor DLBCL 40%

and variants FL ?%
t(11;14)(q13;q32) CCND1 G1 cyclin MCL >95%
MM ~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(8:14)/eBL t(8:14)/sBL
t(11;14) t(3;14) t(4;14)
? t(11;14)

Germinal
Somatic “Class
Hypermutation
Center Switching”

FDC
V(D)J T
recombination Plasma
cell

Naive B
cell apoptosis

apoptosis Memory
B cell

MZL
B-CLL (subset)
ALL MCL BL DLBCL (subset)
FL MM
B-CLL(subtype) DLBCL (subset) 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 • detection of the breakpoint in the

• histological context
• easy for evaluation • needs less patients ’ material
• numerical aberrations, deletions, • control on the presence of the tumor
amplification
population in the sample
• complex patterns
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 Bcl2/IgH MYC BAP MALT1 BAP IGH BAP Bcl1/IgH

samples (%cells with (%cells with (%cells with (%cells with (%cells with
aberrant pattern) aberrant pattern) aberrant pattern) aberrant pattern) aberrant pattern)
mean 11 0,5 4 9,4
SD 2 0,95 1,58 3,4
Cut off 17 4 0 9 20
 FICTION
combined ICH and FISH
CyclinD1
kappa
FICTION Kappa/CyclinD1
Kappa/CyclinD1 BAP Double immunofluorescence
How to integrate the FISH data into histopathology diagnosis?

Genomic abnormality = malignancy

Genomic abnormality = lymphoma type

FL t(14;18)(q32;q21) +
t(14;18)(q32;q21) + (85%) GCB expression profile
bcl2 protein + Bcl2 +/-
favorable prognosis
DLBCL
t(14;18)(q32;q21) –
FL bcl2 protein +
t(14;18)(q32;q21) – ABC expression profile
bcl2 protein +/- 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