Epidemiology of NHL 4% of all cancers 4% of all deaths 8.5 cases / 100.000 / year 65 M:F 1.8 65 higher incidence.

Epidemiology of NHLEpidemiology of NHL 4% of all cancers4% of all cancers 4% of all deaths4% of all deaths 8.5 cases / 100.000 / year <658.5 cases / 100.000 / year <65 69 cases / 100.000 / year >6569 cases / 100.000 / year >65 M:F 1.8 <65 1.3 > 65M:F 1.8 <65 1.3 > 65 higher incidence in Western than higher incidence in Western than

developing countriesdeveloping countries incidence increased 3 fold 1975-95incidence increased 3 fold 1975-95

NHL : etiologic factorsNHL : etiologic factors

Immunodeficiency : primary and acquired Immunodeficiency : primary and acquired (HIV, post-tansplant)(HIV, post-tansplant)

Virus: HTLV-1, EBVVirus: HTLV-1, EBV Helicobacter PyloriHelicobacter Pylori Autoimmune disordersAutoimmune disorders Occupational exposures (pesticides, Occupational exposures (pesticides,

solvents, dyes)solvents, dyes) Other (weak association):Other (weak association): diet (milk, meat), diet (milk, meat),

blood transfusions, familialblood transfusions, familial

Ann Arbor Staging Ann Arbor Staging I: a single lymphatic region or extranodal siteI: a single lymphatic region or extranodal site II: two or more regions on the same side of II: two or more regions on the same side of

diaphragm or one extranodal site and one or diaphragm or one extranodal site and one or more lymphaticmore lymphatic

III: Involvement on both sides of diaphragmIII: Involvement on both sides of diaphragm IV: disseminated to liver, lung, BM, pleura, IV: disseminated to liver, lung, BM, pleura,

bone, skinbone, skin

Diagnostic proceduresDiagnostic procedures History (B symptoms)History (B symptoms) physical examinations (lymph nodes, physical examinations (lymph nodes,

hepatosplenomegaly, Waldeyers ring etc)hepatosplenomegaly, Waldeyers ring etc) Lab.: complete blood count, LDH, bLab.: complete blood count, LDH, b22--

microglobulin, renal and liver functionmicroglobulin, renal and liver function Chest X-ray, abdominopelvic CT scanChest X-ray, abdominopelvic CT scan bilateral BM biopsies and PB smearbilateral BM biopsies and PB smear

Hematopathology Lab.Hematopathology Lab. Processing and diagnosis of bone marrow, Processing and diagnosis of bone marrow,

blood, lymph nodes, tonsils, thymus, spleen blood, lymph nodes, tonsils, thymus, spleen and other tissues with suspect lymphomaand other tissues with suspect lymphoma

Methods:Methods: routine histopathologyroutine histopathology immunohistochemistry on frozen and paraffin immunohistochemistry on frozen and paraffin

sectionssections flow cytometryflow cytometry DNA analysisDNA analysis molecular biologymolecular biology

Routine histopathologyRoutine histopathology

Fixatives:Fixatives: B5 and formalineB5 and formaline StainingsStainings Htx-eosineHtx-eosine GiemzaGiemza PASPAS Gordon-SweetGordon-Sweet

frozen

B5 form.

flow

imprints: DNA

LYMPHOMA LYMPHOMA CLASSIFICATIONSCLASSIFICATIONS

Kiel classification 1974, rev. 1992Kiel classification 1974, rev. 1992 Lukes and Collins classification 1974Lukes and Collins classification 1974 Working Formulation 1984Working Formulation 1984 REAL (Revised European-American REAL (Revised European-American

Classification) Harris et al.Classification) Harris et al.

Blood, 1994, 84, 1361-1392Blood, 1994, 84, 1361-1392

B-cell lymphomasB-cell lymphomas Postulated normal counterparts:Postulated normal counterparts:

stemcell

BM B cellprecursor

B-precursorALL/NHLnullcommonpre-B

AUL

Peripheral B-cells

Lymph nodes

Peripheralblood

Mucosa associatedlymphatic tissue

B-cell lymphomasB-cell lymphomas Postulated normal counterparts:Postulated normal counterparts:• Peripheral B-cellsPeripheral B-cells

small lymphocyte

GC

Mt

Mz

Marginal zone

Mantle zone

FCC

Lymph node

Burkitt?

Large cell NHL

Lpl/ICPC

CLL

CB

CC

HCL??? Proliferating B-cell

recirculating B-cell

Ig producingB-cell

REAL ClassificationREAL Classification

B cell neoplasmsB cell neoplasms I. B-precursor neoplasmsI. B-precursor neoplasms

lymphoblastic leukemia/lymphomalymphoblastic leukemia/lymphoma

II. Peripheral B-cell neoplasmsII. Peripheral B-cell neoplasms

REAL ClassificationREAL Classification II. Peripheral B-cell neoplasmsII. Peripheral B-cell neoplasms 1. B-CLL1. B-CLL 2.Lymphoplasmocytoid lymphoma -2.Lymphoplasmocytoid lymphoma -

immunocytomaimmunocytoma 3.Mantle cell lymphoma3.Mantle cell lymphoma 4.Hairy cell leukemia4.Hairy cell leukemia 5.Plasmacytoma/myeloma5.Plasmacytoma/myeloma

NHL : Flow cytometry NHL : Flow cytometry

Morphology: Lymphocytic lymphomaMorphology: Lymphocytic lymphoma

Immunophenotype: CD19+, kappa+, CD5+, Immunophenotype: CD19+, kappa+, CD5+, CD23+, CD20-, mCD22-, CD10-CD23+, CD20-, mCD22-, CD10-

NHL : Flow cytometryNHL : Flow cytometry ImmunocytomaImmunocytoma Monoclonal k, CD19+, CD20+, CD22+, CD5-, Monoclonal k, CD19+, CD20+, CD22+, CD5-,

CD10-, CD23-CD10-, CD23-

60% B cells,80% B cells CD5- Monocl. kappa

NHL : Flow cytometry NHL : Flow cytometry Morphology: Mantle cell lymphomaMorphology: Mantle cell lymphoma

CD19+ CD5 dim

CD5 dimCD23-

NHL : Flow cytometryNHL : Flow cytometry HAIRY CELL LEUKEMIAHAIRY CELL LEUKEMIA CD19+ cells have characteristic scatter, CD5-, CD19+ cells have characteristic scatter, CD5-,

CD10- (some cases +)CD10- (some cases +)

CD19PE

CD5FITC

NHL : Flow cytometryNHL : Flow cytometry HAIRY CELL LEUKEMIAHAIRY CELL LEUKEMIA CD19+ cells are Bly7+, CD11c+, CD25+CD19+ cells are Bly7+, CD11c+, CD25+

NHL : Flow cytometryNHL : Flow cytometry Myeloma - plasmocytoma:Myeloma - plasmocytoma: CD19-, CD20-, CD22-, CD23-, CD5-, CD19-, CD20-, CD22-, CD23-, CD5-,

CD10-CD10-

R4

CD38 bright,CD45neg CD56+

REAL ClassificationREAL Classification II. Peripheral B-cell neoplasmsII. Peripheral B-cell neoplasms 6. Follicle Center Cell (FCC)6. Follicle Center Cell (FCC) grades: I (small cell), II (mixed small and large cell), III grades: I (small cell), II (mixed small and large cell), III

(large cell)(large cell)

7. Marginal zone B-cell7. Marginal zone B-cell extranodal (MALT +/- monocytoid cells)extranodal (MALT +/- monocytoid cells) nodal (+/- monocytoid cells)nodal (+/- monocytoid cells) splenic marginal zone (+/- villous lymphocytes)splenic marginal zone (+/- villous lymphocytes)

NHL : Flow cytometry NHL : Flow cytometry Morphologic diagnosis : Morphologic diagnosis : Low grade Marginal zone NHLLow grade Marginal zone NHL Triple staining FITC/ PE/ CD20PerCPTriple staining FITC/ PE/ CD20PerCP

R1

64% B cells Monocl.

NHL : Flow cytometry NHL : Flow cytometry Morphologic diagnosis : Morphologic diagnosis : Low grade Marginal zone NHLLow grade Marginal zone NHL Tripple stainings CD23 F/CD5 PE/ CD19TRI and Tripple stainings CD23 F/CD5 PE/ CD19TRI and

CD22 F/CD10PE/CD19 TRICD22 F/CD10PE/CD19 TRI

Most B-cellsexpress CD22 dimand are CD10-

14% B cells CD23+4%B cells CD23+/5+7% of B cells CD5+

Localizations of MALT lymphomasLocalizations of MALT lymphomas conjunctiva inc. orbitconjunctiva inc. orbit salivary glandssalivary glands Waldeyer's ringWaldeyer's ring larynxlarynx thyroid glandthyroid gland breastbreast lunglung GI tractGI tract urogenital tracturogenital tract

NHL : Flow cytometryNHL : Flow cytometry MALT lymphoma, gastric mucosa pxMALT lymphoma, gastric mucosa px B cells were CD20+, CD22+, CD5-, CD10-, B cells were CD20+, CD22+, CD5-, CD10-,

CD23- CD23-

60%B cells

NHL : Flow cytometry NHL : Flow cytometry Morphology: FCC type IIMorphology: FCC type II Partial involvement (confirmed by bcl-2 IH)Partial involvement (confirmed by bcl-2 IH)

R5

45% B-cells

ratio:0,5

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

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

for for

NHL : Flow cytometry NHL : Flow cytometry Morphology: FCC II (CB/CC foll & diff)Morphology: FCC II (CB/CC foll & diff) The medium/large sized cell population is CD10+ and CD22 The medium/large sized cell population is CD10+ and CD22

dim, CD5-, CD23-dim, CD5-, CD23-

REAL ClassificationREAL Classification II. Peripheral B-cell neoplasmsII. Peripheral B-cell neoplasms 8. Diffuse Large B-Cell 8. Diffuse Large B-Cell include various subtypesinclude various subtypes one defined: mediastinal (thymic) B-NHLone defined: mediastinal (thymic) B-NHL

9. Burkitt´s lymphoma9. Burkitt´s lymphoma 10. High-grade Burkitt-like10. High-grade Burkitt-like

NHL : Flow cytometry NHL : Flow cytometry Large cell B-NHL (CB polym. diff.)Large cell B-NHL (CB polym. diff.) Staining CD5F/CD19PE/CD3PerCPStaining CD5F/CD19PE/CD3PerCP CD20-, mCD22-, CD23-, CD10-CD20-, mCD22-, CD23-, CD10- some cells positive for in large cell-gatesome cells positive for in large cell-gate

32% of cells in large-cell gate83% CD19+

NHL : Flow cytometryNHL : Flow cytometry Lymphoblastic lymphoma Burkitt-likeLymphoblastic lymphoma Burkitt-like

R1

L3Scatter

78% B cellsCD19+. CD20dim,m CD22 neg

NHL : Flow cytometryNHL : Flow cytometry

Lymphoblastic lymphoma Burkitt-likeLymphoblastic lymphoma Burkitt-like

CD10+CD22 neg

CD19+, CD5-

T-cell lymphomasT-cell lymphomas Postulated normal counterparts:Postulated normal counterparts:

stemcell

AUL

BMTHYMUS

T ALLcyt.CD3+/TdT+

m3-/4-/8-

m3-/4+/8+

4+ or 8+

T-cellprecursors

Peripheral T-cells

skinMF, SS

Mucosa, bowelIntest. T cell NHL

Lymph node

sinus

Peripheral T NHL

ANLC

REAL ClassificationREAL Classification T cell neoplasmsT cell neoplasms I. Precursor T-cellI. Precursor T-cell lymphoblastic leukemia/lymphomalymphoblastic leukemia/lymphoma

II. Peripheral T cell and NK-cell II. Peripheral T cell and NK-cell neoplasmsneoplasms

REAL ClassificationREAL Classification II. Peripheral T cell and NK-cell II. Peripheral T cell and NK-cell

neoplasmsneoplasms 1. T CLL1. T CLL 2. Large granular lymphocyte (LGL) 2. Large granular lymphocyte (LGL)

leukemialeukemia

T-cell type NK-cell typeT-cell type NK-cell type 3.Mycosis fungoides/Sezary syndrome3.Mycosis fungoides/Sezary syndrome

REAL ClassificationREAL Classification II. Peripheral T cell and NK-cell II. Peripheral T cell and NK-cell

neoplasmsneoplasms 4. Peripheral T cell lymphoma4. Peripheral T cell lymphoma cytologic categories: medium sized, mixed medium cytologic categories: medium sized, mixed medium

and large cell, large cell-lymphoepithelioid celland large cell, large cell-lymphoepithelioid cell 5. Angioimmunoblastic T-cell 5. Angioimmunoblastic T-cell

lymphoma AILDlymphoma AILD 6. Angiocentric lymphoma6. Angiocentric lymphoma

REAL ClassificationREAL Classification II. Peripheral T cell and NK-cell II. Peripheral T cell and NK-cell

neoplasmsneoplasms 7. Intestinal T cell lymphoma7. Intestinal T cell lymphoma (+/- enteropathy associated)(+/- enteropathy associated)

8. Adult T cell lymphoma/leukemia8. Adult T cell lymphoma/leukemia 9.Anaplastic large cell lymphoma9.Anaplastic large cell lymphoma CD30+, T-and null cell typesCD30+, T-and null cell types

NHL : Flow cytometryNHL : Flow cytometry Peripheral T cell NHLPeripheral T cell NHL mCD3-, cytCD3+, CD4+, CD8-,mCD3-, cytCD3+, CD4+, CD8-, CD7+, CD2+,CD5+, HLA-DR neg.CD7+, CD2+,CD5+, HLA-DR neg.

84% T cells mCD3-/CD4+

NHL : Flow cytometryNHL : Flow cytometry Peripheral T cell NHLPeripheral T cell NHL 80% of CD4+ cells were CD45RA, Leu8dim+80% of CD4+ cells were CD45RA, Leu8dim+

gated on CD4/SSC

*TRC V g 1.3 rearrangement

Dermatopatisk lymphadenopathyDermatopatisk lymphadenopathy

CD4/CD8 ratio 15:1CD4/CD8 ratio 15:1 All T cells positive for mCD3, CD5, CD2, CD7All T cells positive for mCD3, CD5, CD2, CD7 Activation of CD4 cellsActivation of CD4 cells

Dermatopatisk lymphadenopathyDermatopatisk lymphadenopathy CD4 positive cells not clonal (no TCR or CD4 positive cells not clonal (no TCR or

rearangement)rearangement) Cd45RA:CD45R0 0,35Cd45RA:CD45R0 0,35 predominance of CD62L+predominance of CD62L+

ImmunohistochemistryImmunohistochemistry

Frozen sections:Frozen sections: skin biopsies, bone marrow biopsiesskin biopsies, bone marrow biopsies APAAP methodAPAAP method Paraffin sectionsParaffin sections lymph nodes and other tumorslymph nodes and other tumors Immunoperoxidase methodImmunoperoxidase method Ventana Ventana

ImmunohistochemistryImmunohistochemistry

Panel of antibodiesPanel of antibodies B T OtherB T Other CD20 CD3 MIB-1CD20 CD3 MIB-1 CD79 UCHL-1 CD45CD79 UCHL-1 CD45 k/l CD43 CD68k/l CD43 CD68 LN1 CD4/CD8 CD30,CD15LN1 CD4/CD8 CD30,CD15

FLow cytometryFLow cytometry

Multiparameter analysisMultiparameter analysis Triple immunofluorescence:Triple immunofluorescence: CD14CD14//GPAGPA//CD45CD45

B TB T lambdalambda//kappakappa//CD20CD20 CD4CD4//CD8CD8//CD3CD3 CD22CD22//CD10CD10//CD20 CD20 CD7CD7//CD2CD2//HLA-DRHLA-DR CD23CD23//CD5CD5//CD19 CD19 CD16CD16//CD56CD56//CD3CD3

Flow cytometryFlow cytometry Other panelsOther panels

T cells Hairy cell leukemiaT cells Hairy cell leukemia

CD25/CD25/CD56CD56//CD3 CD3 CD103/ CD103/CD19CD19//CD20CD20 CD45RA/CD45RA/CD45ROCD45RO//CD4CD4 CD11c/ CD11c/CD19CD19//CD20CD20 CD45RA/CD45RA/CD45ROCD45RO//CD8 CD8 CD25/ CD25/CD19CD19//CD20CD20 CD62L/CD62L/CD4CD4//CD8CD8

Other methodsOther methods

DNA indexDNA index Feulgen staining on imprintsFeulgen staining on imprints PI staining by flowcytometryPI staining by flowcytometry Molecular methodsMolecular methods TCR rearrangementTCR rearrangement Heavy chain rearrangementHeavy chain rearrangement PCR for translocationsPCR for translocations

Chromosomal abnormalities in Chromosomal abnormalities in some NHL entitiessome NHL entities

CLL B tri 12, abn. 13qCLL B tri 12, abn. 13q mantle cell t(11;14) mantle cell t(11;14) bcl-1bcl-1 FCC t(14;18) FCC t(14;18) bcl-2 bcl-2 BurkittBurkitt t(8;14),t(8;22),t(2;8) t(8;14),t(8;22),t(2;8) C-mycC-myc CLL T inv.14(q11;32), tri.8CLL T inv.14(q11;32), tri.8 low grade T tri.3, 5 or +Xlow grade T tri.3, 5 or +X large cell anaplastic t(2;5)large cell anaplastic t(2;5)

Future clinical trialsFuture clinical trials Aggressive lymphomasAggressive lymphomas Dose intensity and dose sizeDose intensity and dose size High dose sequential therapyHigh dose sequential therapy

Indolent lymphomasIndolent lymphomas nucleoside analogs (CDA)nucleoside analogs (CDA) immunotherapyimmunotherapy monoclonal antibodiesmonoclonal antibodies antisense nucleotidesantisense nucleotides

Bone marrow ref.Bone marrow ref.

Tumors of the Bone MarrowTumors of the Bone MarrowBrunning R.D., McKenna R.W.Brunning R.D., McKenna R.W.

Armed Forces Institute of Pathology, Fasc. 9Armed Forces Institute of Pathology, Fasc. 9

Bethesda, MD, 1994Bethesda, MD, 1994

Pathology of Bone MarrowPathology of Bone MarrowNaeim F.Naeim F.

Igakuy-Shoin, New york, Tokyo 1992Igakuy-Shoin, New york, Tokyo 1992

Lymphomas ref.Lymphomas ref. Atlas of Lymphoid Hyperplasia and LymphomaAtlas of Lymphoid Hyperplasia and LymphomaFerry JA., Haris NL.Ferry JA., Haris NL.

WB. SAuders Co, Philadelphia, London etc. 1997WB. SAuders Co, Philadelphia, London etc. 1997

Extranodal LymphomasExtranodal LymphomasIsaacson PG., Norton AJ.Isaacson PG., Norton AJ.

Churchill Livingstone, Edinburgh, London etc., 1994Churchill Livingstone, Edinburgh, London etc., 1994

Thymus, Lymph Nodes, Spleen and LymphaticsThymus, Lymph Nodes, Spleen and LymphaticsHenry K., Symmers W.St C.Henry K., Symmers W.St C.

Churchill Livingstone, Edinburgh, London etc., 1992Churchill Livingstone, Edinburgh, London etc., 1992