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Tools for the Diagnosis of Lymphoproliferative Diseases

When is it difficult to diagnose lymphoproliferative disease?

• Persistent lymphocytosis consisting of small lymphocytes

• Lymph node aspirates containing an excess of small, normal appearing lymphocytes, or intermediate sized, normal appearing lymphocytes

• Rare blasts in the peripheral blood• A pleural effusion containing small

lymphocytes

Peripheral blood Pleural effusion in a cat

Small lymphs

Lymph node aspirate with increased numbers of intermediate sized lymphs

Neoplastic lymphocyte expansion is monoclonal

transformation of a transformation of a single lymphocytesingle lymphocyte

polyclonal responsepolyclonal response

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Flow Cytometry

Antibody binding by fluorescence

CD4

CD

8

Side

scat

ter

Side

scat

ter

(com

plex

ity)

(com

plex

ity)

Forward scatterForward scatter(size)(size)

Light scatter detection Basic markers used to identify lymphocytes

CD21 CD3 CD5CD4 or CD8

B cell T cell

CD34: precursor cells

CD45: pan-leukocyte

Flow cytometry summary

• Flow cytometry can tell you if the lymphocytes in a given population are heterogeneous (a mixture of different types of B and T cells) or homogeneous (all B cells or all a single T cell subpopulation).

• Homogeneous populations of cells are more likely to be neoplastic

Pleural effusions/Mediastinal Masses

Small lymphs

Chylous vs. Lymphoma vs. Thymoma

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Classic Thymoma:Mast cells

Mixed populationof Lymphocytes

-mainly small

*Rarely see neoplasticepithelial cells

Thymoma:

Epithelial cells

Chylous effusion:

CD

8C

D8

CD4CD4

CD

21C

D21

B cell lymphoma:

5/6 recent, confirmed feline mediastinal lymphomas have been B cell, and one a thymic T cell lymphoma

CD

21C

D21

CD

8C

D8

CD4CD4

CD

4

CD8

Thymoma:

“DoublePositive”

Chylous

CD

8C

D8

CD4CD4

Thymic lymphoma-r/o thymoma

CD

8C

D8

CD4CD4Cells slightly larger

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PCR for Antigen Receptor Rearrangement (PARR)

Immunoglobulin gene rearrangement

V genesn = 100 - 200

D genesn = 30

J genesn = 6 Germ Line

Gene within a B cell can vary in size

DNA excisionNucleotide trimmingAddition of nucleotides

Amplification of nonAmplification of non--neoplastic neoplastic lymphoid tissuelymphoid tissue Amplification of lymphomaAmplification of lymphoma

Identification of clonally expanded lymphocyte populations

NegNeg NegNeg B cellB cell

+ ctrlB

cellB

cellT

cell

T cellT cell

Limits of assay detection

100 ng Liver 100 ng spleen

M _ 10% 1% 0.1% 0%10% 1% 0.1%100% 10% 1% 0.1%

neoplastic only

% neoplastic DNA

IgH

The assay detects between 1:100 and 1:1000 neoplastic cells depending

upon the background tissue

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PCR for Antigen Receptor Rearrangement (PARR)

• Sensitivity= 85%– “False Negatives” in 15% of confirmed

lymphomas– Impossible to design primers capable of detecting

all rearrangements

• Specificity= 92%– 8% of PCR+ dogs did not go on to develop

cytologically or histologically confirmed lymphoma during 1 yr of follow-up

Diagnostic gelsNEGATIVE

MONOCLONAL B CELL

Use of the clonality assay to detect early lymphoma

3-8-02: Cytology: Reactive lymphoid hyperplasiaBiopsy: atypical cortical proliferation (concern about the atypia in the cortex, but cannot definitively diagnosis LSA).

March 2002: clinically normal but with mild generalized lymphadenopathy

“Willi” 10 yr MC Golden Retr.

2002 - 2003: clinically normalMay 2003: generalized lymphadenopathy, lethargy, clinical signs.5-3-03: Cytology: LymphomaBiopsy: Lymphoma

Clonal lymphocyte expansions can be detected early

One year

Use of the clonality assay to uncover CLL

4-1-03: Peripheral lymphocytosis (8000 lymphs), most with an LGL morphology4-18-03: Lymphocyte count returned to normal

March 2003: Received vaccine

“Smokey” 8 yr MC MixUndergoing vaccination protocol for unrelated tumor

April - Sept 2003: Tumor in remission9-12-03: Peripheral lymphocytosis, no vaccines for several months11-5-03: Peripheral lymphocytosis before next vaccine treatment

Cytology of LGLs

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Flow cytometry shows that the LGLs express CD8

CD4CD4

CD8

CD8

CD4CD4

CD8

CD8

Normal dogMore CD4+ than CD8+ T cells

PatientMost lymphocytes are CD8+

95% 13%

25%

The LGLs are derived from a clonal T cell population

March, 2003 Nov, 2003

Uses for PCR for lymphoma other than diagnostics

• Stage disease with PCR

• Follow chemotherapy to evaluate efficacy –provides a more objective and quantitative assessment of disease burden

• Follow dogs in remission to determine if we can predict recurrence earlier

Evolution of a B cell lymphoma

LN aspirate

Evolution of a B cell lymphoma

0

5

10

15

TP g

r/dl

Globulins

R

Initial Presentation Out of remission

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Molecular fingerprinting of a B cell tumor

Same sizeSame sequence

Presentation Recurrence

Molecular Remission3/3 T cell lymphomas never

went into PCR remission

Presentation Euth 4 months laterProgressive disease

B N

First day of clinical remission

B NB N

One week tx

B N

Two months

9/10 B cell lymphomas went into PCR remission

Presentation One week tx

Three weeks

Six weeks

Molecular Remission