peripheral blood pleural effusion in a cat · • persistent lymphocytosis consisting of small...
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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