5002-17

2017 WHO Classification-

Hematopoietic & Lymphoid Tumors:
Evolving Concepts, Relevant Testing,
Directed Treatment in Lymphoma:
Part 1 Basic Knowledge
Sonam Prakash, MBBS
University of California, San Francisco

Amy Chadburn, MD
Weill-Cornell Medical College
DISCLOSURE

In the past 12 months, I have not had any

significant financial interest or other relationship
with the manufacturers of the products or
providers of the services that will be discussed in
my presentation.
 Outline
• Handling/processing samples to “rule out
lymphoma”
– Morphology and ancillary studies
• Normal lymph node: morphology and
immunohistochemical stains
• Reactive lymphadenopathies
• B-cell non-Hodgkin lymphoma
• Hodgkin lymphoma
• T-cell lymphoma

3
MULTIPARAMETER APPROACH TO DIAGNOSE
AND CLASSIFY LYMPHOMAS
WHO Classification of Lymphomas
• Requires integration of
– Histopathology
– Immunophenotype:
• Immunohistochemical stains
• Flow cytometric immunophenotyping
– Molecular/cytogenetic features
– Clinical features

5
 Histopathology
• Formalin fixed paraffin-embedded tissue
– Thin sections for embedding: 2-3 mm
– Adequate fixation: 10% formalin
• For 3 mm thick section, a minimum 6 hours of fixation
is needed.
• Fixation is the most important step for evaluation of
lymph node
– Thin (3 or 4 microns) well stained H&E sections

6
 Importance of Thin Sections
THICK SECTIONS THIN SECTIONS

7
 Immunophenotype:
Immunohistochemical stains
• Advantages
– Routinely processed formalin fixed paraffin embedded
tissue
– Large number of antibodies available
– Evaluation of antigenic profile in conjunction with cytology
and growth pattern
– Can be used on necrotic tissue (CD20 is positive on
necrotic B-cells)
• Disadvantages
– Difficult to demonstrate immunoglobulin light restriction
– Inaccurate quantification: T-cell lymphomas
– Certain antibodies cannot be evaluated (e.g. CD103)

8
 Immunophenotype: Flow cytometric
immunophenotyping
• Fresh tissue saved in RPMI
• Advantages:
– Large number of antibodies on a small sample
– Multiple antigens on same cell
– Best way to evaluate surface immunoglobulin light chains: assess for clonality
– Useful to evaluate antigen intensity
– Better quantification of different antigens: particularly useful in T-cell
lymphomas
• Disadvantages
– Requires fresh tissue
– Cannot evaluate architecture
– Loss of cells of interest: Plasma cells and large lymphoid cells
– Certain antibodies cannot be evaluated (e.g. bcl-1)
– Sclerotic or necrotic tissue: insufficient cells
• If imprints are not cellular, unlikely that flow immunophenotyping will be successful

9
 Molecular studies
• Diagnostic/prognostic/therapeutic utility
• Formalin fixed tissue or fresh tissue
• Evaluation of B-cell or T-cell clonality:
– Immunoglobulin or TCR gene rearrangements
• Specific mutations for diagnosis: L265P mutation
in MYD88 gene
• Gene expression profiling: cell of origin in DLBCL
• Next generation sequencing (to be discussed in
part 3)

10
 Cytogenetics/FISH
• Karyotype: Fresh tissue
– Not commonly performed; however can provide
useful diagnostic and prognostic information
– Useful in high grade B-cell lymphomas
• Fluorescence in situ hybridization (FISH)
– Fresh cells or paraffin embedded tissue or touch
imprints
– Diagnosis of certain lymphomas: IGH/BCL2 (follicular
lymphoma); IGH/CCND1 (mantle cell lymphoma); C-
MYC (Burkitt lymphoma); C-MYC, BCL-2 and BCL-6 in
double hit lymphomas
– Prognosis: C-MYC in DLBCL
11
 Handling a Lymph node to Rule Out
lymphomas: Tissue Allocation
• Gross examination of lymph node: size, consistency
• Section the lymph node: fibrous, nodular,
homogenous/fleshy, necrosis
– Sweeping cut through long axis
– Sections no more than 3 mm thick
• Touch imprint:
– Rapid stain (Diff-Quik or H&E)
• Cytology: small or large, monomorphic or heterogeneous, individual
cell characteristics
– Air dried touch imprints: 50:50 mixture of acetone and
methanol for 1 minute (especially useful for FISH, can also be
used for molecular studies like NGS)
• Stored at 20°C, retains most antigens for 1 week
• Stored at -70°C, stable for years

12
 Examples of Touch Imprints
MZL with plasmacytic Anaplastic large
differentiation cell lymphoma

Classical Hodgkin CLL/SLL

lymphoma

13
 Tissue Allocation for Diagnosing
Lymphomas
• Histopathology: Must submit adequate tissue for
morphology, especially if fibrotic or necrotic tissue
• Flow immunophenotyping: Particularly useful for small
B-cell lymphomas and T-cell lymphomas
• Karyotype/FISH on fresh tissue: Institution dependent
• Freeze a small portion (max size 0.5 cm) for possible
molecular studies if adequate tissue available. Store at
-70°C.
• Good histology and adequate formalin fixed paraffin
embedded tissue will enable diagnosis in most cases

14
Tissue Allocation for Diagnosing Lymphomas
Flow Snap Cytogenetics 2-3 mm thick
cytometry freeze sections

1 2 2
2
a b

FORMALIN
Fresh FIXATION Adequate formalin
LN FOR fixation (> 6 hrs)
Good histology HISTOLOGY
Touch
and adequate imprint
formalin fixed
paraffin
embedded
tissue will
enable
diagnosis in
most cases
HISTOPATHOLOGIC EVALUATION OF
LYMPH NODES
 Normal Lymph Node
Hilum
Cortex
Medullary
cords
Medullary
sinus
Paracortex

Subcapsular sinus
Capsule
17
Normal Lymph Node

18
Normal Lymph Node

19
Normal Lymph Node

T-cell Zone Mantle zone Germinal center

20
Normal Germinal Center

21
Monocytoid B-cells

22
 CD20 CD3 CD21

CD10 Bcl-6 Bcl-2

IgD Ki-67

IHC ON NORMAL
LYMPH NODE

23
 Morphologic Patterns in Reactive
Lymph Nodes
• Preservation of architecture with expansion of
a particular compartment
– Follicular
– Paracortical/interfollicular
– Sinusoidal
– Mixed pattern

• Necrosis
• Granulomatous
 FOLLICULAR PATTERN HYPERPLASIA
• Non-specific follicular hyperplasia
• AIDS-type follicular hyperplasia
• Progressive transformation of germinal
centers
• Hyaline-vascular variant Castleman disease
• Follicular hyperplasia with intense
interfollicular plasmacytosis (includes plasma
cell variant Castleman disease)
25
Reactive Follicular Hyperplasia

26
Reactive follicular Follicular lymphoma Reactive FH
hyperplasia (RFH) (FL) • Distinct mantle zone
• Polarized GC cells
(H&E and Ki-67)
• Tingible body
macrophages
• Bcl-2 negative

Follicular lymphoma
• Ill-defined mantle
Bcl-2 Bcl-2 zone
• Monomorphic GC
with loss of
polarization (Ki-67
useful)
• Lack of tingible body
macrophages
• Bcl-2 positive in
~90% cases
Ki-67 Ki-67

27
 Reactive LN in HIV+ Patient
Geographic germinal centers Follicle lysis

28
 AIDS-type follicular hyperplasia
• Characterized by florid follicular hyperplasia
showing:
– Enlarged irregular (“geographic”) germinal centers
– Mantle zone attenuation
– Follicle lysis
• Seen in early AIDS lymphadenopathy
• Can show monotypic surface immunoglobulin
by flow cytometry (clinical history is important
to avoid misdiagnosis of follicular lymphoma)

29
 Primary Follicles
CD20

Primary follicles are Bcl-2 positive!! Be careful to not call them follicular lymphoma.30
Progressive Transformation of
Germinal Centers

31
 Progressive Transformation of
Germinal Centers (PTGC)
• Size at least 4X that of adjacent secondary follicles
• Influx of mantle zone cells into germinal centers
• Often in association with reactive follicular hyperplasia
• Can present as progressively enlarging lymph nodes
• Can be found in biopsies prior to, concurrent with, or
subsequent to a biopsy showing nodular lymphocyte
predominant Hodgkin lymphoma
• Not considered a pre-neoplastic lesion

32
Mantle zone proliferation with
atrophic germinal centers

33
HV Castleman Disease

34
 Hyaline Vascular Castleman Disease
• Typically presents as solitary mass, often in
mediastinum
• Often asymptomatic, or may symptoms due to
compression of adjacent structures
• Cured by surgical excision
• Morphology:
– Reactive follicles with small sclerotic germinal centers,
present throughout node
– Expansion of mantle zones, concentric “onion skin”
appearance
– Frequent penetration of germinal centers by hyalinized
vessels, producing “lollipop” structures
– Vascular proliferation with “hyalinized” vessel walls
present between follicles
35
 FH with interfollicular plasmacytosis
• Characterized by secondary follicles surrounded by
sheets of plasma cells
• Often seen in plasma cell variant of Castleman disease
• Similar features seen in a variety of chronic
inflammatory conditions (e.g., syphilitic lymphadenitis,
rheumatoid lymphadenitis, late AIDS lymphadenitis)
• “Multicentric Castleman disease” is usually plasma cell
type, has associated systemic symptoms, and links with
HHV-8 (100% of HIV+ cases, 50% of HIV- cases)
– Unresponsive to excision

36
HHV-8
37
Paracortical Expansion Pattern
• Nonspecific reactive paracortical hyperplasia
• Viral
• EBV
• CMV
• HSV
• Postvaccinial
• Drug-induced
• Dermatopathic lymphadenitis

38
 Dermatopathic Lymphadenitis
• Marked expansion of interfollicular (paracortical) areas by
T-cells and Langerhans cells (S100+, CD1a+, Langerin+,
CD68+/-)
• Langerhans cells are cytologically benign, and distort but do
not efface nodal architecture
• Brown pigment present in cells (mixture of melanin and
hemosiderin)
• Skin lesion present in region drained by node in almost all
cases
• Morphology can mimic LN involvement by Mycosis
Fungoides (MF). Clinical history is important.
– MF: T-cells show loss of CD7, clonal T-cell receptor gene
rearrangement

39
40
S100 S100

CD1a CD1a

41
 Acute Infectious Mononucleosis
• Adolescent with fever, sore throat,
lymphadenopathy, and enlarged tonsils
• Splenomegaly 50%, hepatomegaly 10%
• Confirm by serologic testing

42
Acute Infectious Mononucleosis

43
Acute Infectious Mononucleosis

EBER

44
 Acute Infectious Mononucleosis
• Immunohistochemical stains
• Expansion is mixture of B and T-cells
• Usually CD8 > CD4
• Many CD30 positive immunoblasts
• EBV positive cells (in situ hybridization)
include both small and large cells

45
Reactive Immunoblastic Proliferation
CD30 CD20

46
 Infectious Mononucleosis Pitfalls
• Immunoblasts resembling R-S cells that are
also CD30 positive can be misdiagnosed as
classical Hodgkin lymphoma
• Increased CD30+, CD20+ immunoblasts can be
misdiagnosed as diffuse large B-cell
lymphoma, especially in cases with necrosis
• Clinical history is extremely important

47
“One should think twice and thrice before
rendering a diagnosis of DLBCL in a patient
younger than 20 years. Infectious
mononucleosis in particular has to be
suspected when … there are many admixed
large T-cells and Waldeyer’s ring is involved.”
ACL Chan & JKC Chan, 2011 Diffuse large
B-cell lymphoma, in Hematopathology (Saunders/Elsevier)

48
 Reactive Lymph Nodes with
Necrosis/apoptosis
• Complete necrosis/infarct
• Kikuchi-Fujimoto lymphadenitis
• Systemic lupus erythematosis related
lymphadenopathy
• Kawasaki disease
Histiocytic necrotizing lymphadenitis

50
Histiocytic Necrotizing Lymphadenitis
(Kikuchi-Fujimoto Disease)
• Isolated, tender, cervical lymphadenopathy present for several months
• Can have mild constitutional symptoms Self-limiting with resolution within
a few months
• Focal to extensive areas of geographic necrosis with admixed karyorrhectic
debris (recognizing morphology is important!)
• Neutrophils, eosinophils, plasma cells are RARE
• Predominance of T-cells (CD8 > CD4 in necrotic phase) with scattered B
immunoblasts
• MPO positive histiocytes / macrophages
• CD123 positive plasmacytoid dendritic cell clusters at the edge of the lesion
• Always suggest SLE if you’re diagnosing Kikuchi-Fujimoto Disease
• Looks very similar to Kikuchi-Fujimoto Disease
• In SLE: Can see basophilic necrotic material, more plasma cells

51
 Reactive Lymph Nodes with a
Sinusoidal Pattern
• Non-specific sinus histiocytosis
• Rosai-Dorfman disease
• Whipple disease
• Exogenous or endogenous lipids
Non-specific sinus histiocytosis

53
Rosai-Dorfman Disease

• Fibrosis
• Histiocytes with
emperipolesis
• Increased plasma cells
• Histiocytes positive for
S-100, CD68, CD163
54
Lymphomas in a sinusoidal pattern
ALCL

CD30

DLBCL

CD20
55
B-CELL LYMPHOMAS

56
 Formulating a Differential Diagnosis
• Based on pattern and cell morphology
• Pattern
– Follicular
– Interfollicular
– Diffuse
• Cytology
– Size
– Nuclear and cytoplasmic features

57
 Histiocyte

Small cells Intermediate cells Large cells

Small round
lymphocyte Centroblast
Lymphoblast

Centrocyte
(Small cleaved)
Burkitt-lymphoma
cell
Immunoblast
Monocytoid small
lymphocyte

Plasmablast
Plasmacytoid small
lymphocyte

Plasma cell

58
 Follicular Lymphoma

Follicular Lymphoma Grading
Grade 1: 0-5 centroblasts per HPF
Grade 2: 6-15 centroblasts per HPF
Grade 3: >15 centroblasts per HPF
Grade 3A: Centrocytes still present • Cytogenetics/FISH: t(14;18)(q32; q21) in ~80%
Grade 3B: Centroblasts only, no
centrocytes
• IHC: B-cells positive for CD20, CD10 (~90% cases),
Bcl-6, Bcl-2 (~90% cases); CD21 positive FDC
meshworks highlighting follicular architecture
• Bcl-2 negative cases: Lack of polarization on
morphology and by Ki-67, monotypic surface
immunoglobulins by flow immunophenotying
cases, Bcl6 rearrangements in ~15% cases
59
Follicular Lymphoma Grade 3B

CD21 CD21

60
 Pediatric Type Follicular Lymphoma
 Mostly children, but some patients up to 25 years
 Mainly nodal disease of head & neck, but tonsil and
testis were next most common sites.
 In contrast to usual follicular lymphoma, cells often
have blastoid morphology, lack of BCL2 protein
expression or translocation
 Localized disease that may not require treatment other
than excision
 Sometimes difficult to distinguish from reactive
follicles; helpful findings to diagnose this lymphoma:
 Flow cytometry: Monotypic immunoglobulin expression
 Ki-67 IHC: Loss of polarization
Ref: Liu et al. Am J Surg Pathol 37: 333; 2013 61
Mantle Cell Lymphoma

• Flow immunophenotyping: CD19+,

CD20+(moderate), CD5+, monotypic sIg
lambda, FMC7+, CD200-, CD23-, CD10-

62
 CD20 CD5

Bcl-1 SOX11 • IHC: Bcl-1+, SOX11+, LEF1-, Bcl-6-

• CD5 can be negative in 10% cases; Bcl-1
can be negative in 5% cases; if CD5+ and
Bcl-1 negative, SOX11 IHC should be done
• Prognostic indicators:
• IHC: Ki-67 >30%*

*Hoster et al. Prognostic Value of Ki-67 Index, Cytology, and

Growth Pattern in Mantle-Cell Lymphoma: Results From
Randomized Trials of the European Mantle Cell Lymphoma
Network.J Clin Oncol. 2016 Apr 20;34(12):1386-94.

63
 Mantle Cell Lymphoma
Blastoid variant Pleomorphic variant

64
 CLL/SLL
Dark areas

• Flow immunophenotyping: CD19+, CD20+(dim),

Pale areas CD5+, CD23+, monotypic sIg(dim), CD200+, CD10-
(Proliferation • IHC: Bcl-1 negative, SOX11 negative, LEF1 positive
• Prognostic indicators:
centers)
• Cytogenetics/FISH:
• Favorable: Del 13q14
• Poor: Del 11q23, 17p-
• Molecular (Blood or marrow):
• Favorable: Mutated Ig Vh genes (CD38-,
ZAP70-)
• Unfavorable: Unmutated Ig Vh genes
(CD38+, ZAP70+) 65
Marginal Zone Lymphoma

Compare with
66
normal T-cell zone
 Marginal Zone Lymphoma
CD20
On IHC: B-cells negative for CD5, CD10,
Bcl-6, Bcl-1 and CD23, positive for Bcl-2.
Flow cytometry: CD19+, CD20+ B-cells
with monotypic surface Ig expression,
negative for CD5, CD10, CD23.
CD3
If flow cytometry is not available: By IHC
may be CD43+ (~50% cases); kappa and
lambda IHC/ISH may show light chain
restriction
Cytogenetics/FISH: MALT-type translocations
not found; trisomy of 3, 8, and 17 have been
reported. Negative for IGH/BCL2 and BCL6
rearrangements 67
 Small B-cell Lymphomas:
Immunophenotype
Common immunophenotype of small B-cell lymphomas
CD20 CD5 CD10 CD23 Bcl-1 Bcl-6 Important points

CLL/SLL + (dim) + - + - - Lack of CD23 or even

CD5 does not
exclude CLL/SLL
Mantle cell + + - - + - CD5 can be negative
lymphoma in 10% cases; Bcl-1
can be negative in
5% cases; if CD5+
and Bcl-1 negative,
do SOX11
Follicular + - + +/- - + CD10 can be
lymphoma negative in ~10%
cases, Bcl-6 is usually
positive, Bcl-2+ in
~90% cases
Marginal zone + - - - - - Can be CD5 positive
lymphoma 68
Diffuse Large B-cell Lymphoma

CD20 CD3
IHC:
• No FDC
meshworks
with CD21
• Negative
for cyclin-
D1

69
 DLBCL: Cell of Origin

GC B-like
DLBCL

Activated B-like
DLBCL

Alizadeh et al.
Nature 403:503;2000
 DLBCL: Cell of Origin (by IHC)
GCB Non-GCB
+ +
CD10 MUM-1
+
_ _
Bcl-6 GCB
_
Non-GCB
• As determined by Han’s algorithm, cell of origin is useful since many clinical trials
are based on this classification
• The intensity of Bcl-6 staining must be moderate to strong
• + = staining in ≥30% of cells

71
Ref: Hans et al. BLOOD, 1 January 2004; vol 103,number 1
 MYC, BCL2, & BCL6 testing in DLBCL
(2016 WHO Classification)
“All large B-cell lymphomas (LBCL) with MYC and BCL2 and/or
BCL6 rearrangements will be included in a single category to
be designated high grade B-cell lymphoma with MYC and
BCL2 and/or BCL6 rearrangements…. A consensus has not
yet been reached to provide specific guidelines as to which
LBCL should have FISH studies. Some believe that all DLBCL
should have genetic studies for the detection of MYC, BCL2,
and BCL6 rearrangements, whereas others would limit them,
for example, to cases with a GCB phenotype and/or high-
grade morphology or to cases with >40% MYC+ cells.”
- Swerdlow et al. Blood: 127: 2375; 2016
72
Detecting Double Hit Protein Positive DLBCL by
IHC
Johnson et al. J Clin Oncol 30:3452; 2012
MYC

IHC Scoring: − Survival difference by IHC score independent

of IPI score, COO subtype, & presence of
▪ MYC+: ≥40% cells true double-hit by FISH
▪ BCL2+: ≥50% cells − High level MYC staining insignificant unless
BCL2 overexpressed as well
73
 Basic Testing in DLBCL
• Cell of origin by IHC
– Can use Han’s algorithm: CD10, Bcl-6, MUM1
• Double protein expression by C-myc (≥40%)
and Bcl-2 (≥50%)
• If resources allow: FISH for C-MYC, BCL2 and
BCL6
– Can be performed on selected cases

74
Burkitt Lymphoma

75
 Burkitt Lymphoma
• CD20+
• CD10+, Bcl6+
• Bcl2-, rare cases may be weakly Bcl-2+
• Ki-67 approaches 100%
• C-MYC (8q24) translocations
– t(8;14), t(2;8), t(8;22)
• A small subset lack C-MYC translocations: may harbor 11q
aberrations*. WHO 2016: New provisional entity
designated Burkitt-like lymphoma with 11q aberration

*Salaverria et al. A recurrent 11q aberration pattern characterizes a subset of MYC-negative high-
grade B-cell lymphomas resembling Burkitt lymphoma. Blood 2014 123:1187-1198

76
 Diagnostic approach to HBCLs. Lymphomas that potentially fall into the HGBL categories can
morphologically resemble B-lymphoblastic leukemia/lymphoma (B-LBL), BL, and DLBCL as well
as lymphomas that are intermediate between DLBCL and BL (DLBCL/BL).

Steven H. Swerdlow et al. Blood 2016;127:2375-2390

©2016 by American Society of Hematology

 B-cell Lymphomas with
Plasmablastic/Anaplastic Morphology

• Plasmablastic lymphoma
• DLBCL, ALK positive

78
 Plasmablastic lymphoma
CD138

C-myc EBER MUM1

CD79a+
Negative for CD20,
PAX5, Cyclin-D1

79
B-cell lymphoid neoplasms that can be
negative for CD45 and CD20
• B-lymphoblastic leukemia/lymphoma
– CD79a or PAX-5 are useful B-cell markers
– TdT and CD34 useful markers to establish immaturity
• Plasmablastic lymphoma:
– CD79a, CD138, kappa, lambda, EBER, MUM-1 are
useful to establish the diagnosis
• DLBCL, ALK+:
– CD79a, CD138, MUM1, kappa, lambda and ALK are
useful to establish the diagnosis

80
HODGKIN LYMPHOMA
Nodular Lymphocyte Predominant
Hodgkin Lymphoma
Classical Hodgkin Lymphoma
 Nodular Lymphocyte Predominant
Hodgkin Lymphoma (NLPHL)

• Elements required for diagnosis

– Compatible morphology
– Compatible immunophenotype
– Compatible clinical presentation
• Isolated lymphadenopathy of long duration
• Cervical, axillary, inguinal
• Can present at extranodal sites: tonsils, parotid, soft
tissue
• Usually no B-symptoms

82
CD20 PAX5 CD21

83
CD3 PD-1

Bcl-6 OCT2

84
Pattern C

Pattern E

Fan et al. Am J Surg Pathol 27:1346; 2003

85
NLPHL, T-cell/histiocyte-rich Pattern
 THRLBCL-like areas in NLPHL cases linked with significantly
increased risk of recurrence/relapse
 THRLBCL-like areas in NLPHL also showed significant increase
in CD163+ histiocytes not seen in typical NLPHL
 At least one study has linked THRLBCL-like areas in NLPHL with
decreased time to progression
 2016 WHO thus now recommends term “THRLBCL-like
transformation of NLPHL” for progression to entirely diffuse
lesions, and noting presence of variant growth patterns in all
cases

 References:
 Fan et al. Am J Surg Pathol 27:1346; 2003
 Xing et al. Blood 123:3567; 2014
86
 Hartmann et al. PLoS One 8:e78812; 2013
 THRLBCL

CD20 CD3

87
CD20 NLPHL
CD20

T/HRBCL

88
 T-cell/histiocyte-rich Large B-cell
Lymphoma (THRLBCL)
• Middle aged males
• High stage disease, poor prognosis
– Fever, malaise, splenomegaly, hepatomegaly
– Lymph nodes, liver, spleen, bone marrow involvement
frequent at the time of diagnosis
• Diffuse architecture
• Scattered large B-cells (at most in small groups)
• >90% small T-cells
• No identifiable component of nodular lymphocyte
predominant Hodgkin lymphoma morphologically or on
immunohistochemical stains
• IHC: CD20+, often CD30-, CD15-, Bcl-6+, Oct2+, EMA+/-
89
 Differential Diagnosis NLPHL Versus THRLBCL

NLPHL THRLBCL
(Nodular lymphocyte predominant Hodgkin lymphoma) (T-cell rich/histiocyte rich large B-cell lymphoma)

Architecture Nodular Diffuse

Neoplastic cells LP (L & H) cells Atypical large cells, can resemble LP

cells or Reed-Sternberg cells

Phenotype CD20+, CD79a+, Oct-2+, CD45+, CD20+, CD79a+, Oct-2+, CD45+,

EMA+/−, CD30−, CD15−, BOB.1+, J EMA+/−, CD30−, CD15−, BOB.1+, EBV−
chain+, EBV−

Background B cells, CD57/PD-1+ cells, T cells, no small B cells, no FDCs, rare

FDC+ (CD21) CD57/PD-1+ cells

Genotype (whole Polyclonal Often monoclonal (B cell)

tissue)

90
Classical Hodgkin Lymphoma

91
CD20 CD3 PAX5 CD3

HRS must be
PAX5 positive

Weak to
variable
CD20 is
acceptable if
other
findings
typical of CHL

Oct-2 and
BOB.1 should
CD30 CD15 MUM1 be negative
to weakly/
variably
positive

CD15 can be
negative in
~20 cases

92
Nodular Pattern Lymphocyte Rich CHL

93
 CHL, Lymphocyte Rich

Germinal Center

94
CD20 CD3

CD21

95
CD20 PAX-5

CD45 CD30 CD45 CD15

96
T-CELL LYMPHOMAS

97
 Why T-cell Lymphomas are Difficult
• Uncommon in the US and Europe
– 5-10% of all non-Hodgkin lymphomas
– Limited data on genetic profile, pathogenesis, treatment
• Many lack consistent morphologic/cytologic features
– Can look like chronic inflammation (Clinical history is
extremely important!)
• Often occur in extranodal sites
• No simple marker of clonality
– Loss of pan T-cell antigens (CD2, CD3, CD5, CD7): Flow
immunophenotyping is very useful
• No well-characterized relation to T-cell developmental
stages

98
 Nodal and Extranodal PTCL
(WHO 2016)
• Nodal T-cell lymphomas
– Anaplastic large cell lymphoma: ALK positive and ALK negative
– T-cell lymphoma with T-follicular helper (TFH) phenotype*
• Angioimmunoblastic T-cell lymphoma
• Follicular T-cell lymphoma
• Nodal PTCL with TFH phenotype
– Peripheral T-cell lymphoma, NOS
• Extranodal T-cell lymphomas
– NK/T-cell lymphoma
– Enteropathy associated T-cell lymphoma
– Hepatopslenic T-cell lymphoma
– Subcutaneous panniculitis like T-cell lymphoma

*The neoplastic cells should express at least 2 or 3 TFH-related antigens,

including CD279/PD1, CD10, BCL6, CXCL13, ICOS, SAP, and CCR5.
Blood. 2016 May 19;127(20):2375-90
99
Anaplastic Large Cell Lymphoma (ALCL)
-Intrasinusoidal
growth pattern
-Cohesive, may
mimic metastatic
carcinoma
-Hallmark
cells/wreath-like
cells/donut
cells/HRS-like cells

100
 CD30 CD4
• -Positive for CD30
(uniform and
strong), CD2, CD5,
CD43, cytotoxic
markers (granzyme-
B, perforin, TIA-1),
EMA, CD4 (usually;
CD2 EMA TIA-1 rare cases CD8+)
• -Variably positive
for CD45
• -Negative for
– CD3, CD7 (usually)
– PAX-5, EBER

101
 Anaplastic Large Cell Lymphoma (ALCL)
ALK positive
ALK • t(2;5)(p23;q35) ALK on chr
2 and NPM on chr 5:
Nuclear and cytoplasmic
positivity
• ALK with other partner
genes:
cytoplasmic/membranous
positivity
• ALK+ ALCL usually seen in
younger patients and
associated with a
favorable prognosis

102
Partial Involvement by ALCL

CD30 ALK

103
 ALCL: Important Points
• Differential diagnosis:
– Hematopoietic neoplasms: Classical Hodgkin lymphoma, PTCL NOS
– Non-hematopoietic: Metastatic carcinoma, melanoma
• Morphology: Hallmark cells always present (but not specific)
• Can look like reactive, can show partial involvement (sinusoidal
growth pattern)
• IHC: Uniform strong staining for CD30
• CD45 may or may not be positive, often negative for CD3
• TCR gene rearrangement: Clonal (90% of cases)

• Brentuximab vedotin can be used for therapy: Anti-CD30 drug

conjugate (anti-CD30 chimeric monoclonal antibody and the potent antimicrotubule drug
monomethylauristatin E)

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 Angioimmunoblastic T-cell Lymphoma (AITL)

• Lymphoma of follicular helper T lymphocytes,

also associated with
– Extrafollicular proliferation of follicular dendritic cells
– EBV-positive B immunoblasts

• Frequently presents with

– Generalized lymphadenopathy
– Systemic symptoms (hepatosplenomegaly, polyclonal
hypergammaglobulinemia, skin rash, hemolytic
anemia and other autoimmune phenomena)
– Aggressive, median survival <3 years

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 Angioimmunoblastic T-cell Lymphoma (AITL):
Histologic Features
• Complete or partial effacement by polymorphous infiltrate of small to
medium-sized lymphocytes with variable (often minimal) nuclear atypia;
frequently with clear cell morphology

• Prominent proliferation of arborizing high endothelial venules

• Marked expansion of follicular dendritic cell meshworks outside the

follicles, frequently wrapping around high endothelial venules

• Background of mixed inflammatory cells (plasma cells, eosinophils,

immunoblasts, rare RS-like cells); residual regressed follicles may be
present

• Frequent extracapsular extension with preservation and distension of

subcapsular sinuses

• Almost always have associated EBV+ immunoblasts (can morphologically

and immunophenotypically resemble Reed-Sternberg cells and be clonal);
can also have associated small monoclonal plasma cell proliferations
106
Angioimmunoblastic T-cell Lymphoma (AITL)

Effaced nodal architecture by a diffuse lymphoid proliferation 107

AITL: Clear cells and high endothelial venules

108
 Angioimmunoblastic T-cell Lymphoma (AITL)

Immunophenotype:

– Neoplastic lymphocytes have follicular helper T cell

immunophenotype (CD3+, CD4+, CD10+, Bcl-6+, PD-
1+, CXCL13+)

– Expanded follicular dendritic cell networks (CD21+,

CD23+, CD35+)

– EBV+ B immunoblasts

109
CD20 CD3 CD4 CD10

PD-1 CD21 EBER

110
 Angioimmunoblastic T-cell Lymphoma (AITL):
Differential Diagnoses
• Reactive lymphadenopathies: cases with preserved nodal
architecture and paracortical expansion

• Multicentric Castleman's disease: cases with significant numbers of

admixed plasma cells and regressed germinal centers

• Diffuse large B-cell lymphoma: cases with increased numbers of B-

cells in the paracortex

• Classical Hodgkin's Disease: cases with Reed-Sternberg like EBV+

cells and admixed inflammatory cells (eosinophils, plasma cells)

• Peripheral T-cell lymphoma, NOS

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 Peripheral T-cell Lymphoma, Not Otherwise Specified
(PTCL, NOS)
• Heterogeneous category of nodal and extranodal mature T-
cell lymphomas, do not correspond to any of the
specifically defined mature T-cell lymphomas

• Usually peripheral lymph node involvement, but any site

can be affected; generalized disease can involve bone
marrow, liver, spleen, extranodal tissues; leukemic
presentation uncommon

• Majority have advanced disease with B symptoms

• Highly aggressive, poor response to therapy, frequent

relapse
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 Peripheral T-cell Lymphoma, Not Otherwise Specified
(PTCL, NOS)

• Paracortical or diffuse infiltrates with effacement of the

normal architecture

• Broad morphologic spectrum, monomorphous to highly

polymorphous.

• Often irregular, pleomorphic, hyperchromatic or vesicular

nuclei, prominent nucleoli, mitotic figures.

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 Peripheral T-cell Lymphoma, Not Otherwise Specified
(PTCL, NOS)

• Immunophenotype of neoplastic T-cells:

 Frequent down regulation of CD5 and CD7; better
evaluated by flow cytometry
 Positive: CD3, CD4 (usually)
 Negative: CD10, BCL-6, PD1, CXCL13
 CD30 and cytotoxic markers are variable

• Clonal TCR gene rearrangement; very useful in difficult

cases

• Often complex karyotype

114
Extranodal NK/T Cell Lymphoma, Nasal
Type
• Predominantly extranodal lymphoma, most commonly
involving upper aerodigestive tract (nasal cavity,
nasopharynx, paranasal sinuses, palate); classically in
adult Asians (M>F);
– often localized to the upper aerodigestive tract at
presentation
• Extranasal presentation can occur (“extranasal NK/T
cell lymphoma”) including skin, soft tissue, GI tract,
gonads; can have secondary lymph node involvement;
rarely primary lymph node involvement;
– Often have high stage disease at presentation
• Strong EBV association

115
Extranodal NK/T Cell Lymphoma, Nasal
Type
• Microscopic:
– Diffuse lymphoid infiltrate with angiocentric and
angiodestructive growth pattern
– Vascular thrombosis and fibrinoid changes,
extensive coagulative necrosis, mucosal ulceration
– Neoplastic lymphocytes can be small, medium or
large (broad cytologic spectrum)
– Can have associated mixed inflammatory infiltrate
– Can have pseuodepitheliomatous hyperplasia of
mucosal epithelium

116
Extranodal NK/T Cell Lymphoma, Nasal
Type
• Immunophenotype:
– Positive: cytoplasmic CD3 (negative for surface
CD3), CD56 (most cases), cytotoxic markers (TIA-
1, perforin, granzyme B), CD2, EBV (EBER and
LMP); +/- CD7 and CD30
• EBV is positive in NK/T-cells and not B-cells
– Negative: surface CD3, CD4, CD8, CD5, TCR-delta
and BF1, CD16, CD57; B cell markers

117
 Extranodal NK/T Cell Lymphoma, Nasal
Type

• Angiocentric
• Necrosis
118
CD3 CD56

TIA-1 EBER

119
 Extranodal NK/T Cell Lymphoma:
Important Points
• Can look like an inflammatory process
• Association with EBV
– EBV is positive in the T-cells
• Angiocentric, angioinvasive process, necrosis +
• Positive for CD3, CD56 (usually), EBV and
cytotoxic markers
– Absence of CD56 does not exclude NK/T cell
lymphoma
• TCR gene rearrangement: mostly not clonal
120
Approach to Diagnosing B-cell Lymphomas
• Clinical history is very important: Age, duration of lymphadenopathy, sites of
involvement, presence/absence of B-symptoms
• H&E:
– Normal or abnormal
• If abnormal
– Reactive (preservation of architecture) or neoplastic
• If neoplastic evaluate the architecture
– Nodular/follicular, interfollicular or diffuse
• Distribution of atypical cells
– Scattered or in sheets
• Cellular morphology
– Monomorphic, pleomorphic
– Size: Small, medium or large
– Shape: Round, cleaved, irregular
– Burkitt morphology or plasmablastic morphology
– Chromatin: Mature or immature
• Mature (large cells): DLBCL, Burkitt lymphoma, blastoid mantle cell lymphoma
(pleomorphic), plasmablastic lymphoma
• Immature: Lymphoblastic lymphoma, blastoid mantle cell lymphoma (classic), Burkitt
lymphoma

121
 Immunohistochemical Stains
• Should be ordered according to the
differential on morphology
• If CD20 negative and CD3 negative
– CD138, Kappa, lambda (Plasmablastic lymphoma,
Plasmablastic myeloma)
– Cyclin-D1 (Plasmablastic myeloma)
– TdT, PAX-5, CD79a (Lymphoblastic lymphoma)
– ALK (ALK positive DLBCL, ALK positive ALCL)
– CD30, CD15 (Classical Hodgkin lymphoma, ALCL)

122
 Ancillary Studies
• Depends on the differential diagnosis based
on morphology
– FISH or molecular studies can be extremely
valuable in difficult cases
– Remember the pitfalls of positive clonality: can be
seen in reactive conditions
– Before ordering stains or other ancillary studies,
think how those results will affect the diagnosis

123
 Acknowledgements

• Dr. Patrick Treseler, UCSF

• Dr. Karthik Ganapathi, UCSF
• Dr. Kristie White, UCSF

• Dr. Amy Chadburn, Weill-Cornell Medical College

• Dr. Mohamed Salama, University of Utah School
of Medicine

124