Epidemiology of NHL
4%
of all cancers
4% of all deaths
8.5 cases / 100.000 / year <65
69 cases / 100.000 / year >65
M:F 1.8 <65 1.3 > 65
higher incidence in Western than
developing countries
incidence increased 3 fold 1975-95
NHL : etiologic factors
 Immunodeficiency
: primary and acquired
(HIV, post-tansplant)
 Virus: HTLV-1, EBV
 Helicobacter Pylori
 Autoimmune disorders
 Occupational exposures (pesticides,
solvents, dyes)
 Other (weak association): diet (milk, meat),
blood transfusions, familial
Ann Arbor Staging
I:
a single lymphatic region or extranodal site
II: two or more regions on the same side of
diaphragm or one extranodal site and one or
more lymphatic
III: Involvement on both sides of diaphragm
IV: disseminated to liver, lung, BM, pleura,
bone, skin
Diagnostic procedures
History
(B symptoms)
physical examinations (lymph nodes,
hepatosplenomegaly, Waldeyers ring etc)
Lab.: complete blood count, LDH, b2microglobulin, renal and liver function
Chest X-ray, abdominopelvic CT scan
bilateral BM biopsies and PB smear
Hematopathology Lab.
 Processing
and diagnosis of bone marrow,
blood, lymph nodes, tonsils, thymus, spleen
and other tissues with suspect lymphoma
 Methods:
 routine
histopathology
 immunohistochemistry on frozen and paraffin
sections
 flow cytometry
 DNA analysis
 molecular biology
Routine histopathology
 Fixatives:
 B5
and formaline
 Stainings
 Htx-eosine
 Giemza
 PAS
 Gordon-Sweet
frozen
B5 form.
imprints: DNA
flow
LYMPHOMA
CLASSIFICATIONS
Kiel
classification 1974, rev. 1992
Lukes and Collins classification 1974
Working Formulation 1984
REAL (Revised European-American
Classification) Harris et al.
Blood, 1994, 84, 1361-1392
B-cell lymphomas
Postulated
stem
cell
AUL
normal counterparts:
BM B cell
precursor
B-precursor
ALL/NHL
null
common
pre-B
Peripheral B-cells
Lymph
nodes
Peripheral
blood
Mucosa associated
lymphatic tissue
B-cell lymphomas
Postulated
normal counterparts:
• Peripheral B-cells
Marginal zone
Lymph
small
lymphocyte node
Mz
Mt
Mantle zone
FCC
CB
B-cell
Burkitt?
CC
HCL???
Ig producing
Lpl/IC
PC
recirculating
B-cell
GC
Proliferating B-cell
Large cell NHL
CLL
REAL Classification
B
cell neoplasms
I.
B-precursor neoplasms
lymphoblastic leukemia/lymphoma
II.
Peripheral B-cell neoplasms
REAL Classification
II.
Peripheral B-cell neoplasms
1. B-CLL
2.Lymphoplasmocytoid lymphoma immunocytoma
3.Mantle cell lymphoma
4.Hairy cell leukemia
5.Plasmacytoma/myeloma
NHL : Flow cytometry
Morphology: Lymphocytic lymphoma
Immunophenotype: CD19+, kappa+, CD5+,
CD23+, CD20-, mCD22-, CD10-
NHL : Flow cytometry
 Immunocytoma
 Monoclonal
k, CD19+, CD20+, CD22+, CD5-,
CD10-, CD2360% B cells,
80% B cells CD5-
Monocl. kappa
NHL : Flow cytometry
 Morphology:
Mantle cell lymphoma

CD19+
CD5 dim
CD5 dim
CD23-
NHL : Flow cytometry
HAIRY CELL LEUKEMIA
CD19+
cells have characteristic scatter, CD5-,
CD10- (some cases +)
CD19PE
CD5FITC
NHL : Flow cytometry
HAIRY CELL LEUKEMIA
CD19+
cells are Bly7+, CD11c+, CD25+
NHL : Flow cytometry
Myeloma
- plasmocytoma:
CD19-, CD20-, CD22-, CD23-, CD5-,
CD10CD38 bright,
CD45neg
R4
CD56+
REAL Classification
II.
Peripheral B-cell neoplasms
6. Follicle Center Cell (FCC)
 grades:
I (small cell), II (mixed small and large cell), III
(large cell)
7. Marginal zone B-cell
 extranodal (MALT +/- monocytoid cells)
 nodal (+/- monocytoid cells)
 splenic marginal zone (+/- villous lymphocytes)
NHL : Flow cytometry
 Morphologic
diagnosis :
 Low grade Marginal zone NHL
 Triple staining
FITC/ PE/ CD20PerCP
64% B cells
R1

Monocl.

NHL : Flow cytometry
 Morphologic
diagnosis :
 Low grade Marginal zone NHL
 Tripple stainings CD23 F/CD5 PE/ CD19TRI and
CD22 F/CD10PE/CD19 TRI
Most B-cells
express CD22 dim
and are CD10-
14% B cells CD23+
4%B cells CD23+/5+
7% of B cells CD5+
Localizations of MALT lymphomas
 conjunctiva
inc. orbit
 salivary glands
 Waldeyer's ring
 larynx
 thyroid gland
 breast
 lung
 GI tract
 urogenital tract
NHL : Flow cytometry
 MALT
lymphoma, gastric mucosa px
 B cells were CD20+, CD22+, CD5-, CD10-,
CD23
60%
B cells
NHL : Flow cytometry
 Morphology:
FCC type II
 Partial involvement (confirmed by bcl-2 IH)
45% B-cells
R5
 ratio:
0,5
NHL : Flow cytometry
 Morphology:
FCC II (CB/CC foll&diff)
 A CD19 dim population was present
NHL : Flow cytometry
 Morphology:
 The
for
FCC II (CB/CC foll&diff)
medium/large sized cell population is monoclonal
NHL : Flow cytometry
 Morphology:
 The
FCC II (CB/CC foll & diff)
medium/large sized cell population is CD10+ and CD22
dim, CD5-, CD23-
REAL Classification
II.
Peripheral B-cell neoplasms
8. Diffuse Large B-Cell
 include
various subtypes
 one defined: mediastinal (thymic) B-NHL
9.
Burkitt´s lymphoma
10. High-grade Burkitt-like
NHL : Flow cytometry
 Large
cell B-NHL (CB polym. diff.)
 Staining CD5F/CD19PE/CD3PerCP
 CD20-, mCD22-, CD23-, CD10 some cells positive for
in large cell-gate
32% of cells
in large-cell gate
83% CD19+
NHL : Flow cytometry
 Lymphoblastic
lymphoma Burkitt-like
78% B cells
CD19+. CD20dim,
m CD22 neg
L3
Scatter
R1

NHL : Flow cytometry
 Lymphoblastic
lymphoma Burkitt-like
CD19+, CD5-
CD10+
CD22 neg
T-cell lymphomas
Postulated
BM stem
cell
normal counterparts:
THYMUS
m3-/4-/8AUL
T-cell m3-/4+/8+
precursors
T ALL 4+ or 8+
cyt.CD3+/TdT+
Peripheral T-cells
skin
MF, SS
Mucosa, bowel
Intest. T cell NHL
Lymph node
Peripheral
T NHL
ANLC
sinus
REAL Classification
T cell neoplasms
I. Precursor T-cell
lymphoblastic
leukemia/lymphoma
II. Peripheral T cell and NK-cell
neoplasms

REAL Classification
II.
Peripheral T cell and NK-cell
neoplasms
1.
T CLL
2. Large granular lymphocyte (LGL)
leukemia
T-cell type
NK-cell type
3.Mycosis fungoides/Sezary syndrome
REAL Classification
II.
Peripheral T cell and NK-cell
neoplasms
4. Peripheral T cell lymphoma
 cytologic
categories: medium sized, mixed medium
and large cell, large cell-lymphoepithelioid cell
5. Angioimmunoblastic
T-cell
lymphoma AILD
6. Angiocentric lymphoma
REAL Classification
II.
Peripheral T cell and NK-cell
neoplasms
7. Intestinal T cell lymphoma
 (+/-
enteropathy associated)
8. Adult
T cell lymphoma/leukemia
9.Anaplastic large cell lymphoma
CD30+, T-and null cell types
NHL : Flow cytometry
 Peripheral
T cell NHL
 mCD3-, cytCD3+, CD4+, CD8-,
 CD7+, CD2+,CD5+, HLA-DR neg.
84% T cells
mCD3-/CD4+
NHL : Flow cytometry
 Peripheral
 80%
T cell NHL
of CD4+ cells were CD45RA, Leu8dim+
gated on
CD4/SSC
*TRC V g 1.3 rearrangement
Dermatopatisk lymphadenopathy
 CD4/CD8
ratio 15:1
 All T cells positive for mCD3, CD5, CD2, CD7
 Activation of CD4 cells
Dermatopatisk lymphadenopathy
 CD4
positive cells not clonal (no TCR
rearangement)
 Cd45RA:CD45R0 0,35
 predominance of CD62L+
or
Immunohistochemistry
 Frozen
sections:
 skin biopsies, bone marrow biopsies
 APAAP method
 Paraffin sections
 lymph nodes and other tumors
 Immunoperoxidase method
 Ventana
Immunohistochemistry
Panel
of antibodies
B
T
Other
CD20
CD3
MIB-1
CD79
UCHL-1
CD45
 k/l
CD43
CD68
LN1
CD4/CD8 CD30,CD15
FLow cytometry
Multiparameter analysis
 Triple
immunofluorescence:
 CD14/GPA/CD45
B
T
 lambda/kappa/CD20 CD4/CD8/CD3
 CD22/CD10/CD20
CD7/CD2/HLA-DR
 CD23/CD5/CD19
CD16/CD56/CD3
Flow cytometry
 Other
panels
T cells




Hairy cell leukemia
CD25/CD56/CD3
CD45RA/CD45RO/CD4
CD45RA/CD45RO/CD8
CD62L/CD4/CD8
CD103/CD19/CD20
CD11c/CD19/CD20
CD25/CD19/CD20
Other methods
DNA index
Feulgen
staining on imprints
PI staining by flowcytometry
Molecular methods
TCR rearrangement
Heavy chain rearrangement
PCR for translocations
Chromosomal abnormalities in
some NHL entities
CLL B
tri 12, abn. 13q
mantle cell t(11;14) bcl-1
FCC
t(14;18) bcl-2
Burkitt t(8;14),t(8;22),t(2;8) C-myc
 CLL T
inv.14(q11;32), tri.8
low grade T tri.3, 5 or +X
large cell anaplastic t(2;5)
Future clinical trials
Aggressive
lymphomas
 Dose
intensity and dose size
 High dose sequential therapy
Indolent
 nucleoside
lymphomas
analogs (CDA)
 immunotherapy
 monoclonal antibodies
 antisense nucleotides
Bone marrow ref.
Tumors
of the Bone Marrow
Brunning R.D., McKenna R.W.
Armed Forces Institute of Pathology, Fasc. 9
Bethesda, MD, 1994
Pathology
of Bone Marrow
Naeim F.
Igakuy-Shoin, New york, Tokyo 1992
Lymphomas ref.
 Atlas of Lymphoid Hyperplasia and
Ferry JA., Haris NL.
WB. SAuders Co, Philadelphia, London etc. 1997
Lymphoma
 Extranodal Lymphomas
Isaacson PG., Norton AJ.
Churchill Livingstone, Edinburgh, London etc., 1994
 Thymus,
Lymph Nodes, Spleen and Lymphatics
Henry K., Symmers W.St C.
Churchill Livingstone, Edinburgh, London etc., 1992