1. Epidemiology of NHL 4% of all cancers 4% of all deaths
8.5 cases / / year <65
69 cases / / year >65
M:F 1.8 < > 65
higher incidence in Western than developing countries
incidence increased 3 fold

2. 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

3. 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

4. Diagnostic procedures
History (B symptoms)
physical examinations (lymph nodes, hepatosplenomegaly, Waldeyers ring etc)
Lab.: complete blood count, LDH, b2-microglobulin, renal and liver function
Chest X-ray, abdominopelvic CT scan
bilateral BM biopsies and PB smear

5. 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

6. Routine histopathology
Fixatives:
B5 and formaline
Stainings
Htx-eosine
Giemza
PAS
Gordon-Sweet
frozen
flow
form. B5
imprints: DNA

7. 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,

8. B-cell lymphomas Postulated normal counterparts: Lymph nodes
Peripheral B-cells
stem
cell
BM B cell
precursor
Lymph
nodes
Peripheral
blood
AUL
B-precursor
ALL/NHL
null
common
pre-B
Mucosa associated
lymphatic tissue

9. B-cell lymphomas Postulated normal counterparts: Peripheral B-cells
Marginal zone
Lymph
node
small
lymphocyte
Mantle zone
Ig producing
B-cell
FCC Mz
Lpl/IC PC
Burkitt? Mt CB CC
recirculating
B-cell GC
HCL???
CLL
Proliferating B-cell
Large cell NHL

10. REAL Classification B cell neoplasms I. B-precursor neoplasms
lymphoblastic leukemia/lymphoma
II. Peripheral B-cell neoplasms

11. 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

12. NHL : Flow cytometry Morphology: Lymphocytic lymphoma
Immunophenotype: CD19+, kappa+, CD5+, CD23+, CD20-, mCD22-, CD10-

13. NHL : Flow cytometry Immunocytoma
Monoclonal k, CD19+, CD20+, CD22+, CD5-, CD10-, CD23-
60% B cells,
80% B cells CD5-
Monocl. kappa

14. NHL : Flow cytometry  Morphology: Mantle cell lymphoma CD19+ CD5 dim

15. NHL : Flow cytometry HAIRY CELL LEUKEMIA
CD19+ cells have characteristic scatter, CD5-, CD10- (some cases +)
CD19PE
CD5FITC

16. NHL : Flow cytometry HAIRY CELL LEUKEMIA
CD19+ cells are Bly7+, CD11c+, CD25+

17. NHL : Flow cytometry Myeloma - plasmocytoma:
CD19-, CD20-, CD22-, CD23-, CD5-, CD10-
CD38 bright,
CD45neg
CD56+

18. 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)

19. NHL : Flow cytometry Morphologic diagnosis :
Low grade Marginal zone NHL
Triple staining  FITC/ PE/ CD20PerCP
64% B cells 
Monocl. 

20. 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+

21. Localizations of MALT lymphomas
conjunctiva inc. orbit
salivary glands
Waldeyer's ring
larynx
thyroid gland
breast
lung
GI tract
urogenital tract

22. NHL : Flow cytometry MALT lymphoma, gastric mucosa px
B cells were CD20+, CD22+, CD5-, CD10-, CD23- 
60%
B cells

23. NHL : Flow cytometry Morphology: FCC type II
Partial involvement (confirmed by bcl-2 IH)
 ratio:
0,5
45% B-cells

24. NHL : Flow cytometry Morphology: FCC II (CB/CC foll&diff)
A CD19 dim population was present

25. NHL : Flow cytometry Morphology: FCC II (CB/CC foll&diff)
The medium/large sized cell population is monoclonal for 

26. NHL : Flow cytometry Morphology: FCC II (CB/CC foll & diff)
The medium/large sized cell population is CD10+ and CD22 dim, CD5-, CD23-

27. 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

28. 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+

29. NHL : Flow cytometry  Lymphoblastic lymphoma Burkitt-like L3 Scatter
78% B cells
CD19+. CD20dim,
m CD22 neg  L3
Scatter

30. NHL : Flow cytometry Lymphoblastic lymphoma Burkitt-like CD19+, CD5-
CD22 neg

31. T-cell lymphomas Postulated normal counterparts: THYMUS
Peripheral T-cells BM
stem
cell
skin
MF, SS
m3-/4-/8-
Mucosa, bowel
AUL
Intest. T cell NHL
Lymph node
T-cell
precursors
m3-/4+/8+
Peripheral
T NHL
T ALL
cyt.CD3+/TdT+
4+ or 8+
ANLC
sinus

32. II. Peripheral T cell and NK-cell neoplasms
REAL Classification
T cell neoplasms
I. Precursor T-cell
lymphoblastic leukemia/lymphoma
II. Peripheral T cell and NK-cell neoplasms

33. 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

34. 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

35. 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

36. NHL : Flow cytometry Peripheral T cell NHL mCD3-, cytCD3+, CD4+, CD8-,
CD7+, CD2+,CD5+, HLA-DR neg.
84% T cells
mCD3-/CD4+

37. NHL : Flow cytometry Peripheral T cell NHL
80% of CD4+ cells were CD45RA, Leu8dim+
gated on
CD4/SSC
*TRC V g 1.3 rearrangement

38. Dermatopatisk lymphadenopathy
CD4/CD8 ratio 15:1
All T cells positive for mCD3, CD5, CD2, CD7
Activation of CD4 cells

39. Dermatopatisk lymphadenopathy
CD4 positive cells not clonal (no TCR or rearangement)
Cd45RA:CD45R0 0,35
predominance of CD62L+

40. Immunohistochemistry
Frozen sections:
skin biopsies, bone marrow biopsies
APAAP method
Paraffin sections
lymph nodes and other tumors
Immunoperoxidase method
Ventana

41. Immunohistochemistry
Panel of antibodies
B T Other
CD CD MIB-1
CD UCHL CD45
k/l CD CD68
LN CD4/CD8 CD30,CD15

42. FLow cytometry Multiparameter analysis Triple immunofluorescence:
CD14/GPA/CD45
B T
lambda/kappa/CD20 CD4/CD8/CD3
CD22/CD10/CD CD7/CD2/HLA-DR
CD23/CD5/CD CD16/CD56/CD3

43. Flow cytometry Other panels T cells Hairy cell leukemia
CD25/CD56/CD CD103/CD19/CD20
CD45RA/CD45RO/CD CD11c/CD19/CD20
CD45RA/CD45RO/CD CD25/CD19/CD20
CD62L/CD4/CD8

44. Other methods DNA index Feulgen staining on imprints
PI staining by flowcytometry
Molecular methods
TCR rearrangement
Heavy chain rearrangement
PCR for translocations

45. 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)

46. Future clinical trials
Aggressive lymphomas
Dose intensity and dose size
High dose sequential therapy
Indolent lymphomas
nucleoside analogs (CDA)
immunotherapy
monoclonal antibodies
antisense nucleotides

47. Bone marrow ref. Tumors of the Bone Marrow Pathology of 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

48. Lymphomas ref. Atlas of Lymphoid Hyperplasia and Lymphoma
Ferry JA., Haris NL.
WB. SAuders Co, Philadelphia, London etc. 1997
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