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ICCS e-Newsletter CSI Winter 2012

This case study explores a 77-year-old male with lymphocytosis, who was found to have clonal CD4+ T-cells expressing cytotoxic markers. The flow cytometric studies revealed a specific TCR-Vbeta region and a distinct phenotypic profile. The findings suggest the possibility of clonal CD4+ T-cell large granular lymphocytosis/leukemia.

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ICCS e-Newsletter CSI Winter 2012

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  1. ICCS e-Newsletter CSI Winter 2012 • Julia Almeida, MD PhD • Department of Medicine and Cancer Research Center • University of Salamanca • Salamanca, Spain

  2. e-CSI – History and physical examination • A 77-year-old male with no significant past medical historywas studied because of a lymphocytosis detected in a routine blood analysis • Patient was asymptomatic • Physical examination did not revealed any pathological sign (no organomegalies, no skin involvement)

  3. e-CSI – Peripheral blood cell counts

  4. e-CSI – CBC differential PB: Peripheralblood

  5. e-CSI – Biochemical parameters

  6. e-CSI – Work-up and evaluation • Peripheral Blood in EDTA was received for evaluation of lymphocytosis • Flow cytometricimmunophenotypingwas performed on this sample, and the results from selected multiple-stained tubes are provided for review:

  7. e-CSI – Flow cytometric (FCM) studies Data acquisition was performed in a FACSCanto II (BDB); data analysis was done with the INFINICYT software (Cytognos SL) Sequential FCM strategy and combinations of fluorochromeMAb used: e-Newsletter CSI Summer 2010 cytPERF: cytoplasmicperforine; cytGRZ: cytoplasmicgranzyme B

  8. e-CSI – Flow cytometric (FCM) studies Step 1: screening tube Exclusion of debris Displayonly CD3+ T cells Selection of T cells Refiningselection of T cells CD4+ gated T cells CD3+ gated T cells CD4+ gated T cells CD4+ gated T cells 94% CD56+/CD4+ T cells Most CD4+ T cells (94%) are CD56+. In addition, they show partialexpression of CD8dim and higher SSC values in comparisonto CD56- CD4+ T cells Thesecellsrepresentaround 67% of total leukocytes 94% CD4+ T cells (CD4/CD8 ratio: 15.5)

  9. e-CSI – Flow cytometric (FCM) studies Step 1: screening tube Conclusion The lymphocytosis is at expense of CD4+ T cells showing cytotoxic-related markers: expression of CD56, partial expression of CD8dim and high SSC values (67% of all WBC; 9.78 x 109 cells/l) Are these expanded cells clonal?

  10. e-CSI – Flow cytometric (FCM) studies Step 2: assessment of T-cell clonality CD3+ livegate Displayonly CD3+ T cells CD3+ livegate Selection of T cells Refiningselection of T cells CD3+ gated T cells CD4+ gated T cells Selection of CD4+ T cells Around 93% of CD4+ T cells expressthesame TCR-Vbetaregion (TCR-Vb13.1+) Thisspecifictube has beenselectedfromthe total panel of theTCR Vbeta Kit IOTest Beta Mark IM3497, which includes a total of 24 MAb against different TCR-Vb regions in a 8-tube format

  11. e-CSI – Flow cytometric (FCM) studies Step 2: assessment of T-cell clonality Conclusion YES, the expanded CD4+ T-cells are clonal, as they express the same TCR-Vbeta region assessed by FCM (TCR-Vb13.1+)

  12. e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them: Normal residual CD4+ T cells Clonal CD4+ T cells Clonal CD4+ T cells are mostly CD7-, CD26- and CD28- and CD2hi

  13. e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them: Clonal CD4+ T cells Normal residual CD4+ T cells Clonal CD4+ T cells show a typicalphenotype of effectorcells: CD45RA+ / CD197 (CCR7)- / CD27- / CD45RO-/+dim

  14. e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them: Clonal CD4+ T cells Normal residual CD4+ T cells Clonal CD4+ T cells are CD5+, CD8-/dim , cytTCL1- and HLADR+heterogeneous

  15. e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them: Clonal CD4+ T cells Normal residual (+someclonal) CD4+ T cells 85% of total CD4+ T cells are CD57+. All CD4+ T cells are CD11c- and CD30-

  16. e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them: Clonal CD4+ T cells Normal residual CD4+ T cells Clonal CD4+ T cellsexpressPerforine and Granzyme B at thecytoplasmiclevel and are mostly CD94- and CD16-

  17. Large Granular Lymphocytosis / Leukemia Large granular lymphocyte (LGL) leukemia is a well-recognized disorder of clonal mature CD8+ T lymphocytes or less frequently natural killer cells; in addition, it has recently been shown that clonal LGL lymphocytosis / leukemia may also derive from CD4+ LGL T cells

  18. TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia Clinical characteristics • These cases usually display an indolent clinical course –although rare cases associated with aggressive disease have also been reported – associated with a significantly lower frequency of cytopeniasthan CD8+ LGL leukemias • Accordingly, diagnosis is usually made from a lymphocytosisdetected in a routine blood analysis (>80%) • However, these patients frequently (30%) show associated neoplasias(particularly B-cell chronic leukemias/lymphomas), so clinical outcome is determined by the associated tumor Lima et al, Am J Pathol 2003

  19. TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia Phenotypic characteristics of clonal cells CD4+/NKa+/CD8-/+dimT cells display relatively high FSC/SSC values and frequentdimreactivityfor CD8, and show a phenotype of activated (CD7-/dim/CD2h/HLADR+) peripheral memory or effector(CD26-/CD27-/CD28-/CCR7- with frequent coexpression of CD45RA and RO) cytotoxic (usually CD57+/CD56+and granzyme B + /perforine + ) T cells in the absence of other Nka markers (i.e. CD16, CD94, CD161) Lima et al, Am J Pathol 2003

  20. TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia 45 40 Normal CD4+ T cells 35 30 Clonal CD4+ LGL T cells 25 20 15 10 5 0 vb1 vb9.1 vb8.1 vb6.7 vb7.1 vb5.2 vb6.1 vb18.1 vb5.1 vb5.3 vb2.1 vb3.1 vb11 vb16.1 vb12.2 vb13.1 vb13.6 vb14.1 vb17.1 vb23.1 vb21.3 vb22.1 vb20 Analysis of the TCR-Vbrepertoirebyimmunophenotype Percentage of cases TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia have a restricted TCR-Vb repertoire with a preferential usage of a few TCR-Vb families; notably, in more than 40% of cases clonal cells are TCR-Vb 13.1+ Lima et al, Am J Pathol 2003

  21. TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia In additiontotherestricted TCR-Vbrepertoire, otherevidencesstronglysupportthefact of theexistence of a commonantigen-drivenorigin • Thereis a clearassociationbetweenthe TCR-Vbrepertoire and the HLA genotype: all TCR-Vb13.1+ cases are HLADR*0701 • HLADR7/TCRVb13.1+cases show a highlyhomogeneous and strikingly similar TCR (highhomologyin theirCDR3) Garrido et al, Blood 2007 ¿Whatisthenature of theantigen? Clonal CD4+ LGL T cells showfunctional response tohCMV Rodriguez-Caballero et al, Blood 2008

  22. TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia Summary Patients with CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis / leukemia show an indolent course of the disease; however, they frequently have a second neoplasiaand clinical outcome is usually determined by the associated tumor Clonal CD4+/NKa+/CD8-/+dim T-cells show a typically activated, cytotoxic phenotype of effector T-LGL cells and a restricted TCR-Vb repertoire, with a preferential usage of a few TCR-Vb families TCR-Vb13.1+ patients display a common HLA-DRB1*0701 genotype and clonal cells express identical motifs in their CDR3-TCR-V sequences, supporting a common antigen-driven origin Clonal T cells usually display response to hCMV, suggesting the potential involvement of hCMV in the ontogeny of CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis / leukemia - - - -

  23. TCRab+/CD4+ Large Granular Lymphocytosis / Leukemia References Lima M, Almeida J, Dos Anjos Teixeira M, et al. TCRalphabeta+/CD4+ large granular lymphocytosis: a new clonal T-celllymphoproliferativedisorder. Am J Pathol. 2003 Aug;163(2):763-71. Garrido P, Ruiz-Cabello F, Bárcena P, et al. Monoclonal TCR-Vbeta13.1+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis: evidenceforanantigen-drivenchronic T-cellstimulationorigin.Blood. 2007 Jun 1;109(11):4890-8. Ghia P, Prato G, Stella S, Scielzo C, Geuna M, Caligaris-Cappio F. Age-dependentaccumulation of monoclonal CD4+CD8+ double positive T lymphocytes in theperipheralblood of theelderly. Br J Haematol. 2007 Dec;139(5):780-90. Rodríguez-Caballero A, García-Montero AC, Bárcena P, et al. Expandedcells in monoclonal TCR- alphabeta+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosisrecognizehCMVantigens.Blood. 2008 Dec 1;112(12):4609-16. Olteanu H, Karandikar NJ, Eshoa C, Kroft SH. Laboratoryfindings in CD4(+) large granular lymphocytoses.Int J LabHematol. 2010 Feb;32(1 Pt 1):e9-16. Sáez-Borderías A, Romo N, Ruiz-Cabello F, et al. Natural killercell receptor expressionreflectsthe role of humancytomegalovirus in thepathogenesis of a subset of CD4+ T-celllarge granular lymphocytosis.HumImmunol. 2011 Mar;72(3):226-8.

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