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Flow Cytometric TCR V Beta Analysis for Determination of T cell Clonality and Its Clinical Application Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory.

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Presentation on theme: "Flow Cytometric TCR V Beta Analysis for Determination of T cell Clonality and Its Clinical Application Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory."— Presentation transcript:

1 Flow Cytometric TCR V Beta Analysis for Determination of T cell Clonality and Its Clinical Application Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA

2 Page 2 Altered expression of pan T cell markers like CD7, CD5, CD3, CD2 Subset restriction CD4+ or CD8+ Increased expression of few markers like CD25, Aberrant Expression of antigens like CD10, CD30, CD103 Helpful in diseases with high tumor cell count Flow cytometric diagnosis of T-CLPD

3 Page 3 Altered expression of pan T-cell markers – Viral infections like IM, CMV, others Subset restriction CD4+ or CD8+ may be seen in Viral infections like IM, HIV, Autoimmune diseases Increased expression of few markers like CD25 - Increased T regulatory cells Aberrant Expression of antigens like CD10, CD30 – limited to few NHL only - CD30+ positive T cells can be activated T cells

4 Page 4 T cell clonality usually established by molecular techniques Confirmation & isolation of clonal cell proliferation Follow up of treatment – with low cell count / partially treated Minimal Residual Disease Low cellularity specimens like CSF, FNA deep LNs/organs staging PB - Sezary cells Morphology and IHC is not typical and helpful LGLL

5 Page 5 Mainly qualitative Time Consuming Needs Specific set up Amplification of background T cells may interfere Clonal peak can not define Cell type and does not help in classification of CLPD Highly Sensitive but less specific for malignancy

6 Page 6 T Cell Receptor complex T cell membrane Science :

7 Page 7 SCIENCE. 272, 21 ;1996: Thymus

8 Page 8 The a TCR – chromosome Vβ genes – 46 are functional Vβ gene segments -26 subfamilies by cDNA -75% sequence homology at the DNA level. Palindromic & random additions and deletions Unique V-D-J = 91 – Vβ segments 24 Vβ segments usage – 70% T cells Each individual TCR-Vβ segment is expressed in only a small percentage of T cells. SCIENCE. 272, 21 ;1996: Immunogenetics. 1995;42(6):451-3

9 Page 9 Clonal T-cell expansions are expected to express a single V domain FCA demonstration of a V restricted population is evidence of a T cell clone There are V -specific antibodies now that recognize 70% of all individual V domains – - TCR V beta Repertoire V restricted T cell populations can be quantitatively monitored during and after therapy

10 Page 10 V V V FITC FITC+PE FITC PE

11 Page 11 The IOTest Beta Mark TCR-Vβ Repertoire kit – Beckman Coulter 24 Vβ-specific antibodies - recognize 70% of all individual Vβ domains 8 tube panel ( 8 X 3 = 24) –3 antibodies in 2 colors (FITC, PE and FITC+PE) Cocktail Mix A Mix B Mix C Mix D Mix E Mix F Mix G Mix H FITC Vβ 3Vβ 16Vβ 20Vβ 8Vβ 12Vβ 21.3Vβ 14Vβ 7.2 PE Vβ 5.3Vβ 9Vβ 18Vβ 13.1Vβ 5.2Vβ 23Vβ 11Vβ 13.2 FITC+PE Vβ 7.1Vβ 17Vβ 5.1Vβ 13.6Vβ 2Vβ 1Vβ 22Vβ 4 PerCP CD3 APC CD4 or CD8*

12 Page 12 TCR Vβ Analysis – selection of T cells

13 Page 13 TCR Vβ Family % V usage in lymphocyte subsets Mean + 2 SD

14 Page 14 Human Immunology. 64, 689–695 (2003).

15 Page 15 ALL CD3+ T cells CD3+/CD4+ T cells CD3+/CD8+ T cells MeanRange MeanRange MeanRange V beta 13.53(Normal ) 3.32(Normal ) 4.24(Normal ) V beta 28.3(Normal ) 9.36(Normal ) 5.43(Normal ) V beta 34.68(Normal ) 4.37(Normal ) 4.44(Normal ) V beta 41.91(Normal ) 2.03(Normal ) 1.9(Normal ) V beta (Normal ) 6.71(Normal ) 3.22(Normal ) V beta (Normal ) 1.33(Normal ) 1.12(Normal ) V beta (Normal ) 1.09(Normal ) 0.92(Normal ) V beta (Normal ) 1.93(Normal ) 3.39(Normal ) V beta (Normal ) 1.12(Normal ) 2.44(Normal ) V beta 84.68(Normal ) 4.81(Normal ) 4.06(Normal ) V beta 93.13(Normal ) 4.07(Normal ) 3.47(Normal ) V beta (Normal ) 0.87(Normal ) 0.92(Normal ) V beta (Normal ) 1.82(Normal ) 1.29(Normal ) V beta (Normal ) 4.03(Normal ) 3.42(Normal ) V beta (Normal ) 2.81(Normal ) 3.34(Normal ) V beta (Normal ) 1.86(Normal ) 1.6(Normal ) V beta (Normal ) 2.59(Normal ) 5.74(Normal ) V beta (Normal ) 0.95(Normal ) 0.8(Normal ) V beta (Normal ) 5.46(Normal ) 5.06(Normal ) V beta (Normal ) 1.92(Normal ) 0.57(Normal ) V beta (Normal ) 2.6(Normal ) 2.31(Normal ) V beta (Normal ) 2.46(Normal ) 2.39(Normal ) V beta (Normal ) 4.26(Normal ) 3.17(Normal ) V beta (Normal ) 0.48(Normal ) 1.34(Normal ) Total 69.95(Normal 69.95)72.25(Normal 72.25) (Normal 66.58)

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17 Page 17 Total V beta usage Normal Direct Clonal Indirect Clonal

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19 Page 19 Suggestions in literature : Expression of a single TCR-Vβ Lima M et al. Am J Pathol. 2001;159: > 40% sensitivity 93% & specificity 80% > 60% sensitivity 81% & specificity 100% Beck RC et al. Am J Clin Pathol. 2003;120: times of UNL Morice WG et al. Am J Clin Pathol. 2004;121: fold or > 50% of the T cells 40% to 49% suggestive > 70% failed to react with panel of Ab B Feng,et al. J Clin Pathol : (1)> 50% of a gated T cell subset; or (2)>10 times above its normal ranges T cell subsets; or (3)>70% of gated T cells failed to react to any of the TCR-Vb antibodies Suggestions in literature : Expression of a single TCR-Vβ Lima M et al. Am J Pathol. 2001;159: > 40% sensitivity 93% & specificity 80% > 60% sensitivity 81% & specificity 100% Beck RC et al. Am J Clin Pathol. 2003;120: times of UNL Morice WG et al. Am J Clin Pathol. 2004;121: fold or > 50% of the T cells 40% to 49% suggestive > 70% failed to react with panel of Ab B Feng,et al. J Clin Pathol : (1)> 50% of a gated T cell subset; or (2)>10 times above its normal ranges T cell subsets; or (3)>70% of gated T cells failed to react to any of the TCR-Vb antibodies

20 Page 20 1 – At what level or % – clonal ? 2 – Gating strategy ? 3 – Can we use it in samples like BM, CSF, FNA ? 4 – Can we use it for MRD ?

21 Page 21 Patients & Samples Total number of patients analyzed for screening 41Peripheral Blood Bone Marrow Asp Total samples - 40 ATLL – 24 CTCL – 07 LGL - 06 PTCL - 03 T-PLL - 01 Total number of patients followed up to monitor MRD and response to treatment 14Peripheral Blood Bone Marrow Asp Total samples - 50 Tembhare P et al. Am J Clin Pathol 2011;135: Concurrent molecular studies for TCRG gene rearrangement by PCR were performed in 36 cases.

22 Page 22 Gating System -1 (GS-1) Gating System - 2 (GS-2)

23 Page 23 We found, Using aberrant phenotype based gating strategy i.e. GS-2 Demonstration of > 50% usage of single V beta domain OR Failure to demonstrate > 70% of immunoreactivity Tembhare P et al. Am J Clin Pathol 2011;135:

24 Page 24 MRD - 61(48 PB, 7 FNA, 4 CSF and 2 BM) Sequential samples - 14 patients - 3 years Short immunophenotypic panels and Single clone specific TCR-Vβ antibody cocktails Number of tumor cells per micro liter of blood Tembhare P et al. Am J Clin Pathol 2011;135:

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27 Page 27 The minimum level of clonal T cells cells/ul Ranged from 0.8 cells/ul - 306,603 cells/ul Median value of 1132 cell/ul

28 Page 28 Case No Specimen Immunophenotypic profile TCR V β Family % of Aberrant T Cells Vβ Restricted Cytology Results CD2CD3CD4CD5CD7CD8CD25CD26CD45CD57 1FNA++/ NA 1FNA+/ Positive 2FNA++++* Suspicious 2FNA+++* Atypical cells 2FNA+++* Positive 2FNA++++* Suspicious 2FNA+++++* Suspicious 3FNA++/ Positive 4FNA++/ Positive 5CSF+/ Negative 7CSF+++/-++* 486.5Atypical cells 7CSF+++/ Atypical cells 7CSF+-+/ Negative 7CSF+++/ Negative 7CSF+++/ Positive Patients: 6 ATLL & 2 PTCL-NOS Tembhare P et al. Am J Clin Pathol Tembhare P et al. Am J Clin Pathol 2012;137:

29 Page 29 Sensitive method for detection of T cell clonality Gating strategy – aberrant immunophenotypes based – superior than T cell subset based. Gating strategy – aberrant immunophenotypes based – superior than T cell subset based. Clonality - > 50% TCR-Vβ subset restriction > 70% negative for all Vβ subsets TCR-Vβ Repertoire kit Clonality - > 50% TCR-Vβ subset restriction > 70% negative for all Vβ subsets TCR-Vβ Repertoire kit Useful in low cellularity specimens such as FNA and CSF Quantitative and highly sensitive MRD evaluation Highly sensitive - treatment response & early relapse

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