Otteson, Gregory E.

AbstractBackgroundMultiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression‐free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter‐laboratory variation in MM MRD reporting.MethodsSeventeen participants from 13 countries performed blinded analysis of the same eight de‐identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re‐analyzed for MRD and other bone marrow cells (Stage 2). Inter‐laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3).ResultsIn Stage 1, participants agreed on MRD+/MRD− status 89% and 68% of the time respectively. Inter‐observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD− status improved to 74%. Better consistency enumerating all parameters among operators resulted in near‐unanimous agreement on sample adequacy.ConclusionUniform flow cytometry data analysis substantially reduced inter‐laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint.

Abstract Objectives Flow cytometric detection of T-cell clonality is challenging, particularly in differential diagnosis of immature T-cell proliferations. Studies have shown utility of TRBC1, in conjunction with other T-cell markers, as reliable means to identify T-cell clonality by flow cytometry. One limitation of surface TRBC1 (sTRBC1) evaluation is it cannot be detected in surface CD3 (sCD3)-negative T cells, such as normal or abnormal immature T-cell precursors. Here, we assess surface and cytoplasmic TRBC1 expression patterns in the differential diagnosis of T-lymphoblastic leukemia/lymphoma (T-ALL) vs normal thymocyte expansions. Methods Forty-three samples containing T-ALL, thymoma, normal thymus, and/or indolent T-lymphoblastic proliferation (i-TLBP), were evaluated. Results All 24 cases with normal thymocytes or i-TLBPs revealed a characteristic and reproducible sCD3/sTRBC1 expression pattern indicative of polytypic T-cell maturation. In contrast, all 19 T-ALLs lacked this polytypic maturation pattern and were either completely negative for sCD3/sTRBC1 or showed a minor sCD3-positive subset with a monotypic TRBC1 expression pattern. Cytoplasmic TRBC1 evaluation in 9 T-ALLs demonstrated a monotypic intracellular TRBC1-positive (n = 4) or TRBC1-negative (n = 5) expression, indicative of clonality. Conclusions Our findings demonstrate flow cytometric evaluation of surface and cytoplasmic TRBC1 expression can aid detection of T-cell clonality and differential diagnosis of immature T-cell proliferations.

SummaryMeasurable residual disease (MRD) assessment by marrow‐based next‐generation flow cytometry (NGF) following autologous stem cell transplantation (ASCT) may lead to false‐negative results due to patchy marrow involvement and extramedullary disease in patients with multiple myeloma. We assessed the value of simultaneous MRD evaluation with NGF and serum matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MASS‐FIX). Of all 61 complete responders who were NGF‐negative for MRD, around day‐100 post ASCT, 59% were MASS‐FIX‐positive. At median follow‐up of 26 months, 69% of MASS‐FIX(+)/NGF(−) patients were alive and progression‐free versus 96% of MASS‐FIX(‐)/NGF(−) patients, P = 0·02. MASS‐FIX, a simple peripheral blood‐based assay complements marrow‐based NGF to accurately prognosticate patients with myeloma.

Abstract Objectives The diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL) is challenging because of overlapping immunophenotypic features with reactive T cells and limitations of T-cell clonality assays. We studied whether adding an antibody against T-cell receptor β constant region 1 (TRBC1) to a comprehensive flow cytometry panel could facilitate the diagnosis of T-LGLL. Methods We added TRBC1 antibody to the standard T-cell and natural killer (NK) cell panel to assess T-cell clonality in 56 T-LGLLs and 34 reactive lymphocytoses. In addition, 20 chronic lymphoproliferative disorder of NK cells (CLPD-NKs) and 10 reactive NK-cell lymphocytoses were analyzed. Results Clonal T cells were detected in all available T-LGLLs by monotypic TRBC1 expression and clonal/equivocal T-cell receptor gene rearrangement (TCGR) studies, compared with only 27% of T-LGLLs by killer-cell immunoglobulin-like receptor (KIR) restriction. Overall, 85% of T-LGLLs had a blood tumor burden greater than 500 cells/µL. Thirty-four reactive cases showed polytypic TRBC1 expression, except for 5 that revealed small T-cell clones of uncertain significance. All CLPD-NKs showed expected clonal KIR expression and negative TRBC1 expression. Conclusions Addition of TRBC1 antibody to the routine flow cytometry assay could replace the TCGR molecular study and KIR flow cytometric analysis to assess clonality, simplifying the diagnosis of T-LGLL.

Abstract Objectives Distinguishing between T-cell and natural killer (NK)–cell neoplasms could be difficult given their overlapping immunophenotype. In this study, we investigated whether a flow cytometry assay with cytoplasmic staining for CD3 could be used for this purpose. Methods Flow cytometry immunophenotyping was performed on 19 surface CD3 (sCD3)–negative mature T-cell neoplasms, 10 sCD3-positive mature T-cell neoplasms, 13 mature NK-cell neoplasms, and 19 normal controls. In addition to routine antibody panels (CD2, sCD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A TCRγ/δ), cytoplasmic staining for a monoclonal CD3 antibody (clone SK7/Leu-4) was assessed in all cases. A molecular study for T-cell receptor (TCR) gene rearrangement and an immunohistochemical study for TCRβ were performed. Results Our data showed all T-cell neoplasms were uniformly positive for cytoplasmic CD3 (cCD3) regardless of sCD3 expression, whereas 85% of NK-cell neoplasms completely lacked cCD3 expression. The 2 cases with classic NK-cell immunophenotype but partial cCD3 expression showed no molecular genetic features of T-cell lineage by TCR gene rearrangement studies. Conclusions Uniform cCD3 positivity and homogeneous cCD3 negativity highly suggest T-cell and NK lineage, respectively. When partial cCD3 expression is encountered, additional confirmatory studies should be pursued for the most accurate lineage assignment.

Abstract Background Flow cytometric detection of T‐cell clonality is challenging. The current available methodology for T‐cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T‐cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells. Methods Here, we used a single TRBC1 antibody, in conjunction with other T‐cell associated markers, to detect T‐cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T‐cell neoplasm and 97 patients with no T‐cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1. Results We examined TRBC1 expression on neoplastic T‐cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T‐cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T‐cell neoplasms, and arbitrarily dissected T‐cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets. Conclusions Single TRBC1 antibody detection of T‐cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.

BackgroundThe diagnosis of T‐cell neoplasms is often challenging, due to overlapping features with reactive T‐cells and limitations of currently available T‐cell clonality assays. The description of an antibody specific for one of two mutually exclusive T‐cell receptor (TCR) β‐chain constant regions (TRBC1) provide an opportunity to facilitate the detection of clonal TCRαβ T‐cells based on TRBC‐restriction.MethodsTwenty patients with mature T‐cell neoplasms and 44 patients without evidence of T‐cell neoplasia were studied. Peripheral blood (51), bone marrow (10), and lymph node (3) specimens were evaluated by 9‐color flow cytometry including TRBC1 (CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1 and/or CD2/CD3/CD4/CD5/CD7/CD8/CD26/CD45/TRBC1). Monophasic TRBC1 expression on any immunophenotypically distinct CD4‐positive or CD8‐positive/TCRγδ‐negative T‐cell subset was considered indicative of clonality.ResultsMonophasic (clonal) TRBC1 expression was identified on immunophenotypically abnormal T‐cells from all 20 patients with T‐cell malignancies (100% sensitivity), including 17 cases with either >97% or <3% TRBC1‐positive events, and three cases with monophasic homogenous TRBC1‐dim expression. All immunophenotypically distinct CD4‐positive and CD8‐positive/TCRγδ‐negative T‐cell subsets from 44 patients without T‐cell malignancies showed the expected mixture of TRBC1‐positive and TRBC‐1‐negative subpopulations (non‐clonal), except for seven patients (16%) with very small CD8‐positive T‐cell subsets exhibiting a monophasic (clonal) pattern.ConclusionInclusion of a single anti‐TRBC1 antibody into a diagnostic T‐cell flow cytometry panel facilitates the rapid identification of T‐cell neoplasms, in addition to small monotypic CD8‐positive subsets of uncertain significance. © 2019 International Clinical Cytometry Society