1 / 31

Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA

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. Flow cytometric diagnosis of T-CLPD. Altered expression of pan T cell markers like CD7, CD5, CD3, CD2

ull
Télécharger la présentation

Prashant R. Tembhare, MD Flow Cytometry Unit Laboratory of Pathology NCI/NIH, USA

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

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

  2. Flow cytometric diagnosis of T-CLPD • 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

  3. Problems in routine FCM • 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. Need of T cell Clonality • 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 T cell clonality usually established by molecular techniques

  5. Limitations of Molecular Techniques 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. T cell Receptor (TCR) complex T cell membrane T Cell Receptor complex Science. 1996. 274: 209-219.

  7. αβ polypeptides formation Thymus SCIENCE. 272, 21 ;1996: 1755-62.

  8. TCR Vβ repertoire The a TCR – chromosome 7. 65 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: 1755-62. Immunogenetics. 1995;42(6):451-3

  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 Expanded T Cell Population - VβAnalysis

  10. Anti-TCR VβAb-fluorochrome for each TCR-Vβ segment - identify a Vβ specific T cell subset Vb 7.1 FITC+PE Vb 5.3 PE PE FITC+PE FITC Vb 3 FITC

  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) TCR Vβ Repertoire kit

  12. TCR Vβ Analysis – selection of T cells

  13. VβRepertoire usage in T cells from reactive lymphoid tissues % Vb usage in lymphocyte subsets Mean + 2 SD TCR Vβ Family

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

  15. TCR VβRepertoire usage in T cells : Normal Range

  16. TCR Vβ Analysis: A case of ATLL

  17. Total V beta usage Indirect Clonal Normal Direct Clonal

  18. TCR Vβ Analysis: A case of PTCL

  19. What levels of expanded T-cell subset as a monoclonal T-cell population? • Suggestions in literature : Expression of a single TCR-Vβ • Lima M et al. Am J Pathol. 2001;159:1861-1868 • > 40% sensitivity 93% & specificity 80% • > 60% sensitivity 81% & specificity 100% • Beck RC et al. Am J ClinPathol. 2003;120:785-794. • 1.6 times of UNL • Morice WG et al. Am J ClinPathol. 2004;121:373-383. • 10-fold or > 50% of the T cells • 40% to 49% suggestive • > 70% failed to react with panel of Ab • B Feng,et al. J ClinPathol 2010 63: 141-146. • > 50% of a gated T cell subset; or • >10 times above its normal ranges T cell subsets; or • >70% of gated T cells failed to react to any of the TCR-Vb antibodies

  20. Questions for TCR - V beta analysis 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. NCI Study – 2007 to 2010 Patients & Samples Concurrent molecular studies for TCRG gene rearrangement by PCR were performed in 36 cases. Tembhare P et al. Am J ClinPathol 2011;135:890-900.

  22. Gating Strategy Gating System -1 (GS-1) Gating System -2 (GS-2)

  23. Cut off levels for T cells Clonality • 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 ClinPathol 2011;135:890-900.

  24. TCR-Vβ Monitoring of MRD T NHL • 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 ClinPathol 2011;135:890-900.

  25. Follow up

  26. Number of cells at each interval

  27. The minimum level of clonal T cells - 0.8 cells/ul Ranged from 0.8 cells/ul - 306,603 cells/ul Median value of 1132 cell/ul MRD by TCR-Vβ repertoire

  28. TCR-Vβ Monitoring in Paucicellular specimens FNAs and CSF: • Patients: 6 ATLL & 2 PTCL-NOS Tembhare P et al. Am J ClinPathol2012;137:220-226

  29. Summary Sensitive method for detection of T cell clonality • 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 Useful in low cellularity specimens such as FNA and CSF Quantitative and highly sensitive MRD evaluation Highly sensitive - treatment response & early relapse

More Related