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Single-Antibody Detection of T-Cell Receptor Beta Chain Monotypia Resolves Uncertainties in the Identification and Quantitation of Sézary Cells By Routine Flow Cytometry: Towards Accurate and Unequivocal Blood Staging of Cutaneous T-Cell Lymphomas

BACKGROUND: Sézary syndrome and mycosis fungoides are two clinically distinct neoplasias of CD4-positive skin-resident T-cells that share remarkable morphologic and immunophenotypic similarities, commonly referred together as cutaneous T-cell lymphoma (CTCL). Prognostic clinical staging in CTCL requires flow cytometric quantitation of peripheral blood tumor cells (Sézary cells), based on aberrant loss of CD7 and/or CD26 within the CD4-positive T-cell compartment. Unfortunately, similar T-cell populations are frequently encountered in reactive conditions, creating uncertainty in the diagnostic interpretation. We recently reported a flow cytometric strategy to assess T-cell clonality using a single antibody (JOVI-1) against one of 2 mutually exclusive T-cell receptor beta constant (TRBC) regions randomly selected during T-cell receptor gene rearrangement. We hereby applied this strategy to a diagnostic Sézary cell flow cytometry panel, resulting in rapid, accurate and unequivocal identification and quantitation of clonal CD4-positive T-cells.
 METHODS: We studied 33 peripheral blood specimens from 24 patients with CTCL, and 28 specimens from patients with no clinical or laboratory evidence of T-cell malignancy. A routine Sézary cell flow cytometry panel (CD2/CD3/CD4/CD5/CD7/CD8/CD26/CD45) was modified to include a fluorescent labeled antibody against TRBC1. Gated CD4 T-cells were studied to identify discrete subsets lacking CD7, CD26 or both, in addition to other immunophenotypic abnormalities. Within each subset, clonality was defined as a dominant TRBC1-positive or TRBC1-negative population greater than 85%, or a dominant abnormal TRBC1-dim population, as previously described.
 RESULTS: Peripheral blood specimens from patients with CTCL showed subsets of CD4-positive T-cells lacking CD7 (median=35%; range 1.6% to 96%) or CD26 expression (median=54%; range 6.6% to 98%). Fifteen (45%) of these specimens had other identifiable phenotypic abnormalities, including decreased CD2, CD3, CD4 or CD5. Patients without a T-cell malignancy also had CD4-positive T-cells lacking CD7 (median=13%; range 0.5% to 38%) or CD26 expression (median=9.4%; range 0.7% to 30%), albeit to a lesser extent compared to CTCL patients (p<0.01 and p<0.0001 for CD7 and CD26, respectively). One febrile infant showed loss of CD2 on a small T-cell subset. Discrete CD4-positive T-cell subsets with monotypic TRBC expression were identified in 24 specimens (73%) from patients with CTCL (Figure A), ranging from 30 to12,000 cells/µL (median=632). Importantly, no TRBC-restricted CD4-positive T-cells were detected in any of the 28 patients without a T-cell malignancy. Nine specimens from CTCL patients without a TRBC-restricted subset were reviewed based on morphologic, immunophenotypic and clinical features, yielding no definitive evidence of Sézary cells. Absolute numbers of TRBC-restricted T-cells correlated with numbers of Sézary cells estimated based on lack of CD7 or CD26 expression (R=0.99), or absence of both antigens (R=0.7) (Figure B). However, all cases with no detectable clonal TRBC-restricted T-cells showed significant absolute numbers of CD4-positive T-cells lacking CD7 or CD26 (mean=208 cells/µL, range=22 to 1,078 cells/µL), resulting in common diagnostic uncertainties based on quantitation of these subsets. A narrower gating strategy on these same patients based on the combined loss of CD7 and CD26 yielded lower numbers of abnormal cells (mean=51 cells/µL, range=2 to 556 cells/µL), but grossly underestimated the number of TRBC-restricted CD4-positive T-cells by more than 500 cells/µL in 6 patients with CTCL (Figure B).
 CONCLUSIONS: A single anti-TRBC1 antibody added to a routine Sézary cell flow cytometry assay provides a rapid, simple and low-cost approach to query for clonality within immunophenotypically distinct CD4 T-cell subsets, eliminating the need for a separate T-cell clonality assay. Diagnostic uncertainties and quantitative biases resulting from immunophenotypic overlaps between Sézary cells and reactive T-cells are virtually resolved. Direct quantitation of clonal CD4 T-cells based on TRBC1 restriction is a biologically sound strategy for blood staging in CTCL, effectively overcoming the limitations and uncertainties of staging by immunophenotypic analysis based on current guidelines.
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 Horna: MorphoSys AG: Research Funding.
