T-CELL RECEPTOR (TCR)-VΒ REPERTOIRE FLOW CYTOMETRY FOR T-CELL LYMPHOPROLIFERATIVE DISORDER: A RETROSPECTIVE ANALYSIS OF A SINGLE-CENTER REAL-LIFE LABORATORY EXPERIENCE
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Keywords
T-cell lymphoproliferative disorders, Clonality, Multiparametric flow-cytometry, TCR rearrangements, TCR-Vβ repertoire MFC analysis, Vβ repertoire analysis
Abstract
Background: Discrimination between clonal and reactive cell proliferation is critical for the correct management of T-cell lymphocytosis. Multiparametric flow cytometry (MFC) represents a valuable tool, particularly because it allows to evaluate the T-cell receptor (TCR) Vβ repertoire to pinpoint eventual clonality in T-cell lymphocytosis. A restricted expansion of a single out of the 24 evaluable families or a “clonogram-off” pattern are highly suggestive of the presence of a clonal T-cell population. However, data available on the concordance between MFC TCR-Vβ repertoire and molecular analysis of TCR gene rearrangements, which is regarded as the gold standard for assessing T-cell clonality, are limited.
Objective and methods: We performed a retrospective monocentric study including 307 patients referred to our center for lymphocytosis between 2003 and 2024. The aim of our study was to investigate the diagnostic accuracy of MFC TCR-Vβ repertoire analysis and to compare its performance with molecular analysis of TCR gene rearrangements, for the identification of T-cell clonality.
Results: MFC TCR-Vβ repertoire analysis demonstrated a restricted expansion of a single Vβ family in 67.5% of cases, while clonality in the remaining 32.5% was inferred by a “clonogram-off” pattern. For 215 (70%) patients both MFC TCR-Vβ repertoire analysis and molecular analysis of TCR gene rearrangements were available, showing an absolute concordance (215 out of 215 cases, 100%) between the two methods.
Conclusion: MFC TCR-Vβ repertoire analysis is a rapid, cheap, sensitive, and reliable tool to identify clonal T-cell lymphocytosis. It represents an absolute valid first-line diagnostic approach, and it should be included into the routinely laboratory work-up performed MFC analysis for peripheral lymphocytosis.
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References
[2] Venturi V., Kedzierska K., Turner S.J., Doherty P.C., Davenport M.P. “Methods for comparing the diversity of samples of the T cell receptor repertoire.” J Immunol Methods. 2007 Apr 10;321(1-2):182-95. doi: 10.1016/j.jim.2007.01.019. Epub 2007 Feb 21. PMID: 17337271.
[3] Yazdanifar M., Barbarito G., Bertaina A., Airoldi I. “ γδ T Cells: The Ideal Tool for Cancer Immunotherapy.” Cells. 2020 May 24;9(5):1305. doi: 10.3390/cells9051305. PMID: 32456316; PMCID: PMC7290982.
[4] Kroft S.H., Harrington A.M. “Flow Cytometry of B-Cell Neoplasms.” Clin Lab Med. 2017 Dec;37(4):697-723. doi: 10.1016/j.cll.2017.07.001. Epub 2017 Aug 31. PMID: 29128065.
[5] Seegmiller A.C., Hsi E.D., Craig F.E. “The current role of clinical flow cytometry in the evaluation of mature B-cell neoplasms.” Cytometry B Clin Cytom. 2019 Jan;96(1):20-29. doi: 10.1002/cyto.b.21756. Epub 2018 Dec 13. PMID: 30549186.
[6] Böttcher S., Ritgen M., Kneba M. “Flow cytometric MRD detection in selected mature B-cell malignancies.” Methods Mol Biol. 2013; 971:149-74. doi: 10.1007/978-1-62703-269-8_9. PMID: 23296963.
[7] Craig J.W., Dorfman D.M. “Flow Cytometry of T cells and T-cell Neoplasms.” Clin Lab Med. 2017 Dec;37(4):725-751. doi: 10.1016/j.cll.2017.07.002. Epub 2017 Oct 3. PMID: 29128066.
[8] Mahe E., Pugh T., Kamel-Reid S. “T cell clonality assessment: past, present and future.” J Clin Pathol. 2018 Mar;71(3):195-200. doi: 10.1136/jclinpath-2017-204761. Epub 2017 Oct 21. Erratum in: J Clin Pathol. 2018 Jul;71(7):660. doi: 10.1136/jclinpath-2017-204761corr1. PMID: 29055897; PMCID: PMC5868531.
[9] Mendoza H., Tormey C.A., Rinder H.M., Howe J.G., Siddon A.J. “The utility and limitations of B- and T-cell gene rearrangement studies in evaluating lymphoproliferative disorders.” Pathology. 2021 Feb;53(2):157-165. doi: 10.1016/j.pathol.2020.09.024. Epub 2020 Dec 25. PMID: 33358756.
[10] Beck R.C., Stahl S., O'Keefe C.L., Maciejewski J.P., Theil K.S., Hsi E.D. “Detection of mature T-cell leukemias by flow cytometry using anti-T-cell receptor V beta antibodies.” Am J Clin Pathol. 2003 Nov;120(5):785-94. doi: 10.1309/835B-04QX-GNNF-NRJU. PMID: 14608907.
[11] Peruzzi B., Bencini S., Caporale R. “TCR Vβ Evaluation by Flow Cytometry.” Methods Mol Biol. 2021; 2285:99-109. doi: 10.1007/978-1-0716-1311-5_8. PMID: 33928546.
[12] Lima M., Almeida J., Santos A.H., dos Anjos Teixeira M., Alguero M.C., Queirós M.L., Balanzategui A., Justiça B., Gonzalez M., San Miguel J.F., Orfão A. “Immunophenotypic analysis of the TCR-Vbeta repertoire in 98 persistent expansions of CD3(+)/TCR-alphabeta(+) large granular lymphocytes: utility in assessing clonality and insights into the pathogenesis of the disease.” Am J Pathol. 2001 Nov;159(5):1861-8. doi: 10.1016/s0002-9440(10)63032-5. PMID: 11696446; PMCID: PMC1867049.
[13] Feng B., Jorgensen J.L., Hu Y., Medeiros L.J., Wang S.A. “TCR-Vbeta flow cytometric analysis of peripheral blood for assessing clonality and disease burden in patients with T cell large granular lymphocyte leukaemia.” J Clin Pathol. 2010 Feb;63(2):141-6. doi: 10.1136/jcp.2009.069336. PMID: 20154036.
[14] Langerak A.W., van Den Beemd R., Wolvers-Tettero I.L., Boor P.P., van Lochem E.G., Hooijkaas H., van Dongen J.J. “Molecular and flow cytometric analysis of the Vbeta repertoire for clonality assessment in mature TCRalphabeta T-cell proliferations.” Blood. 2001 Jul 1;98(1):165-73. doi: 10.1182/blood. v98.1.165. PMID: 11418476.
[15] Morice W.G., Kimlinger T., Katzmann J.A., Lust J.A., Heimgartner P.J., Halling K.C., Hanson C.A. “Flow cytometric assessment of TCR-Vbeta expression in the evaluation of peripheral blood involvement by T-cell lymphoproliferative disorders: a comparison with conventional T-cell immunophenotyping and molecular genetic techniques.” Am J Clin Pathol. 2004 Mar;121(3):373-83. doi: 10.1309/3A32-DTVM-H640-M2QA. PMID: 15023042.
[16] Qiu Z.Y., Shen W.Y., Fan L., Wang L., Yu H., Qiao C., Wu Y.J., Lu R.N., Qian J., He G.S., Xu W., Li J.Y. “Assessment of clonality in T-cell large granular lymphocytic leukemia: flow cytometric T cell receptor Vβ repertoire and T cell receptor gene rearrangement.” Leuk Lymphoma. 2015 Feb;56(2):324-31. doi: 10.3109/10428194.2014.921297. Epub 2014 Jul 17. PMID: 24828862.
[17] Hsieh Y.C., Chang S.T., Huang W.T., Kuo S.Y., Chiang T.A., Chuang S.S. “A comparative study of flow cytometric T cell receptor Vβ repertoire and T cell receptor gene rearrangement in the diagnosis of large granular lymphocytic lymphoproliferation.” Int J Lab Hematol. 2013 Oct;35(5):501-9. doi: 10.1111/ijlh.12041. Epub 2013 Jan 3. PMID: 23286298.
[18] van Dongen J.J., Lhermitte L., Böttcher S., Almeida J., van der Velden V.H., Flores-Montero J., Rawstron A., Asnafi V., Lécrevisse Q., Lucio P., Mejstrikova E., Szczepański T., Kalina T., de Tute R., Brüggemann M., Sedek L., Cullen M., Langerak A.W., Mendonça A., Macintyre E., Martin-Ayuso M., Hrusak O., Vidriales M.B., Orfao A.; EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708). “ EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes.” Leukemia. 2012 Sep;26(9):1908-75. doi: 10.1038/leu.2012.120. Epub 2012 May 3. PMID: 22552007; PMCID: PMC3437410.
[19] Orfao A., Matarraz S., Pérez-Andrés M., Almeida J., Teodosio C., Berkowska M.A., van Dongen J.J.M.; EuroFlow. “Immunophenotypic dissection of normal hematopoiesis.” J Immunol Methods. 2019 Dec; 475:112684. doi: 10.1016/j.jim.2019.112684. Epub 2019 Oct 30. PMID: 31676343.
[20] van Dongen J.J., Langerak A.W., Brüggemann M., Evans P.A., Hummel M., Lavender F.L., Delabesse E., Davi F., Schuuring E., García-Sanz R., van Krieken J.H., Droese J., González D., Bastard C., White H.E., Spaargaren M., González M., Parreira A., Smith J.L., Morgan G.J., Kneba M., Macintyre E.A. “ Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.” Leukemia. 2003 Dec;17(12):2257-317. doi: 10.1038/sj.leu.2403202. PMID: 14671650.
[21] Langerak A.W., Szczepański T., van der Burg M., Wolvers-Tettero I.L., van Dongen J.J. “Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations.” Leukemia. 1997 Dec;11(12):2192-9. doi: 10.1038/sj.leu.2400887. PMID: 9447840.
[22] Brochet X., Lefranc M.P., Giudicelli V. “IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis.” Nucleic Acids Res. 2008 Jul 1;36(Web Server issue): W503-8. doi: 10.1093/nar/gkn316. Epub 2008 May 24. PMID: 18503082; PMCID: PMC2447746.
[23] Lefranc M.P., Giudicelli V., Ginestoux C., Bodmer J., Müller W., Bontrop R., Lemaitre M., Malik A., Barbié V., Chaume D. “IMGT, the international ImMunoGeneTics database.” Nucleic Acids Res. 1999 Jan 1;27(1):209-12. doi: 10.1093/nar/27.1.209. PMID: 9847182; PMCID: PMC148137.
[24] Sáez-Cirión A., Lacabaratz C., Lambotte O., Versmisse P., Urrutia A., Boufassa F., Barré-Sinoussi F., Delfraissy J.F., Sinet M., Pancino G., Venet A.; Agence Nationale de Recherches sur le Sida EP36 HIV Controllers Study Group. “HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype.” Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6776-81. doi: 10.1073/pnas.0611244104. Epub 2007 Apr 11. PMID: 17428922; PMCID: PMC1851664.
[25] Viallard J.F., Blanco P., André M., Etienne G., Liferman F., Neau D., Vidal E., Moreau J.F., Pellegrin J.L. “CD8+HLA-DR+ T lymphocytes are increased in common variable immunodeficiency patients with impaired memory B-cell differentiation.” Clin Immunol. 2006 Apr;119(1):51-8. doi: 10.1016/j.clim.2005.11.011. Epub 2006 Jan 18. PMID: 16413828.
[26] Viallard J.F., Bloch-Michel C., Neau-Cransac M., Taupin J.L., Garrigue S., Miossec V., Mercie P., Pellegrin J.L., Moreau J.F. “HLA-DR expression on lymphocyte subsets as a marker of disease activity in patients with systemic lupus erythematosus.” Clin Exp Immunol. 2001 Sep;125(3):485-91. doi: 10.1046/j.1365-2249.2001.01623.x. PMID: 11531958; PMCID: PMC1906149.
[27] Sidorova Y.V., Sychevskaya K.A., Chernova N.G., Julhakyan H.L., Smirnova S.J., Ryzhikova N.V., Gorodetskiy V.R., Naumova E.V., Sudarikov A.B. “High Incidence of Clonal CD8+ T-cell Proliferation in Non-malignant Conditions May Reduce the Significance of T-cell Clonality Assay for Differential Diagnosis in Oncohematology.” Clin Lymphoma Myeloma Leuk. 2020 Apr;20(4):203-208. doi: 10.1016/j.clml.2019.12.021. Epub 2020 Jan 8. PMID: 32046930.
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