Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "ICCS e-Newsletter CSI Winter 2012 Julia Almeida, MD PhD Department of Medicine and Cancer Research Center University of Salamanca Salamanca, Spain."— Presentation transcript:

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

2 A 77-year-old male with no significant past medical history was 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) e-CSI – History and physical examination

3 e-CSI – Peripheral blood cell counts CBCNormal range WBC: 14.6 x 10 9 /l (4.5 – 10.8) RBC: 4.35 x /l(4.0 – 5.4) Hgb: 13.0 g/dl(12.0 – 18.0) Hct: 38.3 %(35.0 – 52.0) MCV: 88.1 fl(80.0 – 99.0) MCH: 29.9 pg(27.0 – 32.0) MCHC: 33.9 g/dl(31.5 – 36.0) RDW: 14.3%(10.5 – 14.5) Plts: 346 x 10 9 /l(150 – 450)

4 e-CSI – CBC differential CBC differential (PB) % from WBC Absolute numbers (x10 9 /l) Reference range (Absolute #) Neutrophils x 10 9 /l Monocytes – 0.9 x 10 9 /l Lymphocytes – 3.5 x 10 9 /l Eosinophils – 0.5 x 10 9 /l Basophils – 0.1 x 10 9 /l PB: Peripheral blood

5 e-CSI – Biochemical parameters ParameterValueNormal range B2 microglobulin (mg/dl)1.0<2.5 mg/l LDH (IU/l)340< 460 IU/l

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

7 e-Newsletter CSI Summer 2010 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) Pac.BPac.OFITCPE PerCP- Cy5.5 PECY7APCAPCH7 Step 1 (screening tube) CD45-CD8 Anti-sIg CD56 Anti-sIg CD4 CD19 CD20CD3- Step 2 (T-cell clonality study) CD4- TCR-V 8 + TCR-V 13.6 TCR-V TCR-V 13.6 CD3-CD8- Step 3 (T-cell CLPD panel) CD4 CD45 CD7 CD27 CD5 CD57 cytPERF CD26 CD197 CD25 CD30 cytGRZ CD3 CD2 CD45RO HLADR - CD16 CD28 CD45RA cytTCL1 CD11c CD94 CD8 Sequential FCM strategy and combinations of fluorochrome MAb used: cytPERF: cytoplasmic perforine; cytGRZ: cytoplasmic granzyme B

8 94% CD4+ T cells (CD4/CD8 ratio: 15.5) 94% CD56+/CD4+ T cells e-CSI – Flow cytometric (FCM) studies Step 1: screening tube Exclusion of debris Selection of T cells Refining selection of T cells Display only CD3+ T cells CD3+ gated T cells CD4+ gated T cells Most CD4+ T cells (94%) are CD56+. In addition, they show partial expression of CD8 dim and higher SSC values in comparison to CD56- CD4+ T cells These cells represent around 67% of total leukocytes

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 CD8 dim and high SSC values (67% of all WBC; 9.78 x 10 9 cells/l) Are these expanded cells clonal?

10 e-CSI – Flow cytometric (FCM) studies Step 2: assessment of T-cell clonality CD3+ live gate CD3+ gated T cells CD3+ live gate Selection of T cells Refining selection of T cells Display only CD3+ T cells Selection of CD4+ T cells CD4+ gated T cells Around 93% of CD4+ T cells express the same TCR-Vbeta region (TCR-V ) This specific tube has been selected from the total panel of the TCR Vbeta Kit IOTest Beta Mark IM3497, which includes a total of 24 MAb against different TCR-V 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-Vb )

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: Pac.BPac.OFITCPE PerCP- Cy5.5 PECY7APCAPCH7 Step 3: T-cell CLPD panel TUBE1CD4CD45CD7CD26CD3CD2CD28CD8 Normal residual CD4+ T cells Clonal CD4+ T cells Clonal CD4+ T cells are mostly CD7 -, CD26 - and CD28 - and CD2 hi

13 e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization Normal residual CD4+ T cells Clonal CD4+ T cells Clonal CD4+ T cells show a typical phenotype of effector cells: CD45RA + / CD197 (CCR7) - / CD27 - / CD45RO -/+dim Pac.BPac.OFITCPE PerCP- Cy5.5 PECY7APCAPCH7 Step 3: T-cell CLPD panel TUBE2CD4CD45CD27CD197CD3CD45ROCD45RACD8 After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:

14 e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization Normal residual CD4+ T cells Clonal CD4+ T cells Clonal CD4+ T cells are CD5 +, CD8 -/dim, cytTCL1 - and HLADR + heterogeneous Pac.BPac.OFITCPE PerCP- Cy5.5 PECY7APCAPCH7 Step 3: T-cell CLPD panel TUBE3CD4CD45CD5CD25CD3HLADRcytTCL1CD8 After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:

15 e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization Normal residual (+some clonal) CD4+ T cells Clonal CD4+ T cells 85% of total CD4+ T cells are CD57 +. All CD4+ T cells are CD11c - and CD30 - Pac.BPac.OFITCPE PerCP- Cy5.5 PECY7APCAPCH7 Step 3: T-cell CLPD panel TUBE4CD4CD45CD57CD30CD3-CD11cCD8 After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:

16 e-CSI – Flow cytometric (FCM) studies Step 3: phenotypic characterization Normal residual CD4+ T cells Clonal CD4+ T cells Clonal CD4+ T cells express Perforine and Granzyme B at the cytoplasmic level and are mostly CD94 - and CD16 - Pac.BPac.OFITCPE PerCP- Cy5.5 PECY7APCAPCH7 Step 3: T-cell CLPD panel TUBE5CD4CD45cytPERFcytGRZCD3CD16CD94CD8 After applying the same gating strategy as previously shown to identify CD4+ T cells, then we proceed to characterize them:

17 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 Large Granular Lymphocytosis / Leukemia

18 TCR +/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 cytopenias than CD8+ LGL leukemias Accordingly, diagnosis is usually made from a lymphocytosis detected 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 TCR +/CD4+ Large Granular Lymphocytosis / Leukemia Phenotypic characteristics of clonal cells T-cell related antigens: CD3+ TCR + CD4++ CD8-/+ CD5+ CD7-/+ CD2++ NK/cytotoxic-cell associated (Nka) markers: CD56+ CD57+ cyGranzyme B+ cyPerforine + CD11b-/+ CD11c- CD16- CD94- CD161- CD158a- NKB1- Cytokine-receptors and activation-associated markers: CD25-CD122-CD38-HLADR+ Maturation-associated antigens: CD197 (CCR7) -CD45RA+ CD45RO+ CD28- CD26- CD27- CD62L- CD4 + /NKa + /CD8 -/+dim T cells display relatively high FSC/SSC values and frequent dim reactivity for CD8, and show a phenotype of activated (CD7 -/dim /CD2 h /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 Analysis of the TCR-V repertoire by immunophenotype TCR +/CD4+ Large Granular Lymphocytosis / Leukemia Normal CD4+ T cells Clonal CD4+ LGL T cells Percentage of cases TCR +/CD4+ Large Granular Lymphocytosis / Leukemia have a restricted TCR- V repertoire with a preferential usage of a few TCR-V families; notably, in more than 40% of cases clonal cells are TCR-V Lima et al, Am J Pathol 2003

21 TCR +/CD4+ Large Granular Lymphocytosis / Leukemia In addition to the restricted TCR-V repertoire, other evidences strongly support the fact of the existence of a common antigen-driven origin There is a clear association between the TCR-V repertoire and the HLA genotype: all TCR-V cases are HLADR*0701 HLADR7/TCRV cases show a highly homogeneous and strikingly similar TCR (high homology in their CDR3) ¿What is the nature of the antigen? Clonal CD4+ LGL T cells show functional response to hCMV Rodriguez-Caballero et al, Blood 2008 Garrido et al, Blood 2007

22 Patients with CD4 + /NKa + /CD8 -/+dim T-LGL lymphocytosis / leukemia show an indolent course of the disease; however, they frequently have a second neoplasia and 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-V repertoire, with a preferential usage of a few TCR-V families TCR-V 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 TCR +/CD4+ Large Granular Lymphocytosis / Leukemia Summary

23 TCR +/CD4+ Large Granular Lymphocytosis / Leukemia References 1.Lima M, Almeida J, Dos Anjos Teixeira M, et al. TCRalphabeta+/CD4+ large granular lymphocytosis: a new clonal T-cell lymphoproliferative disorder. Am J Pathol Aug;163(2): TCRalphabeta+/CD4+ large granular lymphocytosis: a new clonal T-cell lymphoproliferative disorder. 2.Garrido P, Ruiz-Cabello F, Bárcena P, et al. Monoclonal TCR-Vbeta13.1+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis: evidence for an antigen-driven chronic T-cell stimulation origin. Blood Jun 1;109(11): Monoclonal TCR-Vbeta13.1+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis: evidence for an antigen-driven chronic T-cell stimulation origin. 3.Ghia P, Prato G, Stella S, Scielzo C, Geuna M, Caligaris-Cappio F. Age-dependent accumulation of monoclonal CD4+CD8+ double positive T lymphocytes in the peripheral blood of the elderly. Br J Haematol Dec;139(5): Age-dependent accumulation of monoclonal CD4+CD8+ double positive T lymphocytes in the peripheral blood of the elderly. 4.Rodríguez-Caballero A, García-Montero AC, Bárcena P, et al. Expanded cells in monoclonal TCR- alphabeta+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis recognize hCMV antigens. Blood Dec 1;112(12): Expanded cells in monoclonal TCR- alphabeta+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis recognize hCMV antigens. 5.Olteanu H, Karandikar NJ, Eshoa C, Kroft SH. Laboratory findings in CD4(+) large granular lymphocytoses. Int J Lab Hematol Feb;32(1 Pt 1):e9-16.Laboratory findings in CD4(+) large granular lymphocytoses. 6.Sáez-Borderías A, Romo N, Ruiz-Cabello F, et al. Natural killer cell receptor expression reflects the role of human cytomegalovirus in the pathogenesis of a subset of CD4+ T-cell large granular lymphocytosis. Hum Immunol Mar;72(3):226-8.Natural killer cell receptor expression reflects the role of human cytomegalovirus in the pathogenesis of a subset of CD4+ T-cell large granular lymphocytosis.


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

Similar presentations


Ads by Google