Horna, Pedro

Abstract Objectives Prognostically relevant chromosomal abnormalities in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) are routinely identified by fluorescence in situ hybridization (FISH) on peripheral blood or bone marrow specimens. We studied the prevalence of chromosomal abnormalities on extramedullary tissues involved by CLL/SLL and evaluated their association with prominent proliferation centers (PPCs). Methods FISH for recurrent abnormalities in CLL/SLL was performed on formalin-fixed, paraffin-embedded biopsy sections. PPCs were identified on H&E-stained sections. Available FISH results on peripheral blood or bone marrow specimens were also reviewed. Results Recurrent FISH abnormalities were detected in 69% of 320 CLL/SLL biopsy specimens studied, including +12 (35%), 13q– (24%), 11q– (15%), 17p– (6%), 6q– (2%), and IGH/BCL2 (0.9%). Forty-three patients had abnormal blood or bone marrow FISH analyses, of whom 7 (16%) had discordant +12 and/or 13q–, and 3 (7%) had discordant 17p– or 11q–. Morphology was positive (17%), negative (78%), or equivocal (6%) for PPCs on 247 evaluable biopsy specimens, a finding not significantly associated with FISH results (P = .7). Conclusions Trisomy 12 is overrepresented in tumoral CLL/SLL involvement, compared with the known predominance of 13q– in blood. Discrepancies between leukemic and tissue FISH findings are occasionally encountered. FISH results do not correlate with the presence of PPCs.

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.

T-cell clonality testing is integral to the diagnostic work-up of T-cell malignancies; however, current methods lack specificity and sensitivity, which can make the diagnostic process difficult. The recent discovery of a monoclonal antibody (mAb) specific for human TRBC1 will greatly improve the outlook for T-cell malignancy diagnostics. The anti-TRBC1 mAb can be used in flow cytometry immunophenotyping assays to provide a low-cost, robust, and highly specific test that detects clonality of immunophenotypically distinct T-cell populations. Recent studies demonstrate the clinical utility of this approach in several contexts; use of this antibody in appropriately designed flow cytometry panels improves detection of circulating disease in patients with cutaneous T-cell lymphoma, eliminates the need for molecular clonality testing in the context of large granular lymphocyte leukemia, and provides more conclusive results in the context of many other T-cell disorders. It is worth noting that the increased ability to detect discrete clonal T-cell populations means that identification of T-cell clones of uncertain clinical significance (T-CUS) will become more common. This review discusses this new antibody and describes how it defines clonal T-cells. We present and discuss assay design and summarize findings to date about the use of flow cytometry TRBC1 analysis in the field of diagnostics, including lymph node and fluid sample investigations. We also make suggestions about how to apply the assay results in clinical work-ups, including how to interpret and report findings of T-CUS. Finally, we highlight areas that we think will benefit from further research.

AbstractIntroducing a sensitive and specific peripheral blood flow cytometric assay for Sézary syndrome and mycosis fungoides (SS/MF) requires careful selection of assay design characteristics, and translation into a laboratory developed assay through development/optimization, validation, and continual quality monitoring. As outlined in a previous article in this series, the recommended design characteristics of this assay include at a minimum, evaluation of CD7, CD3, CD4, CD8, CD26, and CD45, analyzed simultaneously, requiring at least a 6 color flow cytometry system, with both quantitative and qualitative components. This article provides guidance from an international group of cytometry specialists in implementing an assay to those design specifications, outlining specific considerations, and best practices. Key points presented in detail are: (a) Pre‐analytic components (reagents, specimen processing, and acquisition) must be optimized to: (i) identify and characterize an abnormal population of T‐cells (qualitative component) and (ii) quantitate the abnormal population (semi/quasi‐quantitative component). (b)Analytic components (instrument set‐up/acquisition/analysis strategy and interpretation) must be optimized for the identification of SS/MF populations, which can vary widely in phenotype. Comparison with expert laboratories is strongly encouraged in order to establish competency. (c) Assay performance must be validated and documented through a validation plan and report, which covers both qualitative and semi/quasi‐quantitative assay components (example template provided). (d) Ongoing assay‐specific quality monitoring should be performed to ensure consistency.

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.

AbstractThis review discusses the definition and major categories of cutaneous T‐cell lymphoma, Sézary syndrome and mycosis fungoides, and the role of immunophenotyping in their diagnosis. The following key points are raised: (a) Sézary syndrome and mycosis fungoides cells most often have a characteristic CD3+ CD4+ CD7− and/or CD26− immunophenotype. (b) This immunophenotype is not specific, but can assist in the distinction from non‐neoplastic T cells and other subtypes of mature T‐cell neoplasm. (c) However, small subsets of normal and reactive T‐cells can have an overlapping immunophenotype, and can be distinguished by evaluating for additional changes in antigen expression.

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.