Tools for the Diagnosis of Lymphoproliferative Diseases When is it difficult to diagnose lymphoproliferative disease? Persistent lymphocytosis consisting of small Lymph node aspirates containing an excess of small, normal appearing, or intermediate sized, normal appearing Rare blasts in the peripheral blood A pleural effusion containing small Peripheral blood Pleural effusion in a cat Small lymphs Lymph node aspirate with increased numbers of intermediate sized lymphs eoplastic lymphocyte expansion is monoclonal polyclonal response transformation of a single lymphocyte 1
Antibody binding by fluorescence Flow Cytometry Light scatter detection asic markers used to identify Side scatter (complexity) Forward scatter (size) cell CD21 CD34: precursor cells 5: pan-leukocyte T cell CD3 CD5 or Flow cytometry summary Pleural effusions/mediastinal Masses Flow cytometry can tell you if the in a given population are heterogeneous (a mixture of different types of and T cells) or homogeneous (all cells or all a single T cell subpopulation). Homogeneous populations of cells are more likely to be neoplastic Small lymphs Chylous vs. Lymphoma vs. Thymoma 2
Classic Thymoma: Mast cells Thymoma: Mixed population of Lymphocytes -mainly small Epithelial cells *Rarely see neoplastic epithelial cells Chylous effusion: cell lymphoma: CD21 CD21 5/6 recent, confirmed feline mediastinal lymphomas have been cell, and one a thymic T cell lymphoma Thymoma: Chylous Thymic lymphoma-r/o thymoma Double Positive Cells slightly larger 3
PCR for Antigen Receptor Rearrangement (PARR) Immunoglobulin gene rearrangement V genes n = 100-200 D genes n = 30 J genes n = 6 Germ Line DA excision ucleotide trimming Addition of nucleotides Gene within a cell can vary in size Amplification of non-neoplastic neoplastic lymphoid tissue Amplification of lymphoma Identification of clonally expanded lymphocyte populations T cell cell cell + ctrl Limits of assay detection % neoplastic DA M _ 100% 10% 1% 0.1% 10% 1% 0.1% 10% 1% 0.1% 0% IgH neoplastic only 100 ng Liver 100 ng spleen eg eg cell T cell The assay detects between 1:100 and 1:1000 neoplastic cells depending upon the background tissue 4
PCR for Antigen Receptor Rearrangement (PARR) Sensitivity= 85% False egatives in 15% of confirmed lymphomas Impossible to design primers capable of detecting all rearrangements Diagnostic gels EGATIVE MOOCLOAL CELL Specificity= 92% 8% of PCR+ dogs did not go on to develop cytologically or histologically confirmed lymphoma during 1 yr of follow-up Use of the clonality assay to detect early lymphoma Clonal lymphocyte expansions can be detected early Willi 10 yr MC Golden Retr. March 2002: clinically normal but with mild generalized lymphadenopathy 3-8-02: Cytology: Reactive lymphoid hyperplasia iopsy: atypical cortical proliferation (concern about the atypia in the cortex, but cannot definitively diagnosis LSA). 2002-2003: clinically normal May 2003: generalized lymphadenopathy, lethargy, clinical signs. 5-3-03: Cytology: Lymphoma iopsy: Lymphoma One year Use of the clonality assay to uncover CLL Smokey 8 yr MC Mix Undergoing vaccination protocol for unrelated tumor Cytology of LGLs March 2003: Received vaccine 4-1-03: Peripheral lymphocytosis (8000 lymphs), most with an LGL morphology 4-18-03: Lymphocyte count returned to normal April - Sept 2003: Tumor in remission 9-12-03: Peripheral lymphocytosis, no vaccines for several months 11-5-03: Peripheral lymphocytosis before next vaccine treatment 5
Flow cytometry shows that the LGLs express The LGLs are derived from a clonal T cell population 95% 13% 25% Patient Most are + ormal dog More + than + T cells March, 2003 ov, 2003 Uses for PCR for lymphoma other than diagnostics Stage disease with PCR Evolution of a cell lymphoma 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 L aspirate Evolution of a cell lymphoma Initial Presentation Out of remission 15 TP gr/dl 10 5 Globulins 0 R 6
Molecular fingerprinting of a cell tumor Presentation Recurrence Molecular Remission 3/3 T cell lymphomas never went into PCR remission Presentation One week tx First day of clinical remission Two months Euth 4 months later Progressive disease Same size Same sequence Molecular Remission 9/10 cell lymphomas went into PCR remission Presentation One week tx Three weeks Six weeks 7