A practical approach to risk stratification of incidental T-cell clonality

Abstract

Because T-cell neoplasms often present with nonspecific findings, T-cell clonality assessment is performed across many clinical scenarios, including the workup of cytopenias, lymphocytosis, eosinophilia, and suspected lymphoma. Sensitive assays, including flow cytometric staining of the T-cell receptor (TCR) constant region and molecular-based TCR clonality testing, have enhanced our ability to detect T-cell neoplasms, but these techniques frequently identify T-cell clones in patients without suspicion of T-cell malignancy. These incidentally detected clones, sometimes called T-cell clones of uncertain significance, do not clearly have universal potential for progression to overt T-cell neoplasia. However, their detection can prompt unnecessary diagnostic procedures, generate unwarranted patient anxiety, and even lead to inappropriate therapeutic interventions. Here, we propose a practical framework for risk stratification of unexpected T-cell clones in peripheral blood that minimizes the risk of unnecessary intervention while maintaining vigilance for true T-cell malignancy.

Introduction

T-cell neoplasms are heterogeneous disorders with diverse clinical presentations and outcomes. Because they often present with nonspecific clinical findings, accurate diagnosis requires careful integration of clinical, morphologic, immunophenotypic, and molecular data. A defining diagnostic feature of T-cell neoplasia is the demonstration of clonal T-cell receptor (TCR) gene rearrangements, reflecting the expansion of a single T-cell population. Advances in clonality testing, particularly molecular-based assays and flow cytometric detection of TCR β-chain constant region (TRBC1/2) expression, have improved the sensitivity of identifying clonal T-cell populations and are increasingly used by clinicians and pathologists in the diagnosis and monitoring of T-cell neoplasms (Figure 1; Box 1).

Figure 1.

Pathophysiologic mechanisms underlying detection of T-cell clonality by flow cytometry or molecular methods. T-cell neoplasia is initiated when a T cell acquires a molecular or cytogenetic abnormality that drives cellular proliferation and/or survival (top). In the period of time before diagnostic criteria for a T-cell neoplasm entity are met, or before the T-cell clone becomes clinically apparent, the clone may still be detectable by sensitive techniques such as flow cytometry or molecular techniques. Nonneoplastic mechanisms may also drive detectable T-cell clonality (bottom). These include robust clonal or oligoclonal expansion in the setting of antigen stimulation, such as in infection or autoimmunity, as well as homeostatic proliferations in the setting of lymphopenia. Clones generated by these mechanisms do not have inherent malignant or premalignant potential. There is emerging but limited evidence that, in the setting of sufficiently robust and persistent antigen stimulation, T-cell clones may acquire pathogenic mutations. However, the frequency of such acquisitions remains unclear. Here, we define a TCUS as any T-cell clone detectable by flow cytometry or molecular techniques that does not meet diagnostic criteria for a T-cell neoplasm, thereby, encompassing both neoplastic and nonneoplastic mechanisms of T-cell clonality. Figure created with biorender.com. Wilk, A. (2026) https://app.biorender.com/illustrations/canvas-beta/6a430b640cb10096131bea9c.

Box 1. Defining and detecting T-cell clonality.

Flow cytometry: commercially available reagents have been developed that detect expression of the constant regions of the TCRβ chain, that is, TRBC1 and TRBC2. Because inclusion of TRBC1 or TRBC2 is required for successful rearrangement and expression of an αβ TCR, and because the selection of TRBC1 or TRBC2 during rearrangement is random and mutually exclusive, staining for TRBC1/2 can be used to detect clonal T-cell populations by flow cytometry.^56,68, 69, 70, 71, 72, 73 Antibodies against both TRBC1 and TRBC2 are available commercially and may be used either in combination or individually. For instance, a flow cytometry panel that includes CD3, TCRγδ, and TRBC1 may be sufficient to assess T-cell clonality, because a surface CD3⁺ population that is negative for both TCRγδ and TRBC1 may be inferred to be TRBC2 restricted.^55,56 Several guidelines have proposed using an 85% threshold to define a T-cell clone; that is, an αβ T-cell population that is >85% TRBC1⁻ or TRBC1⁺.^55,73 However, clinical laboratories differ in the implementation of this cutoff. Laboratories may use bulk-gating strategies, identifying a clone only when the total T-cell repertoire is >85% TRBC1⁻ or TRBC1⁺. Other laboratories use difference-from-normal gating strategies, first identifying a T-cell subpopulation with atypical cell-surface marker expression, followed by examining whether the candidate subpopulation is >85% TRBC1⁻ or TRBC1⁺.⁵⁵ The latter strategy enables more sensitive detection of T-cell clones, including small clones that may be of uncertain significance. There are 2 important caveats to interpreting TRBC staining by flow cytometry. First, because anti-TRBC1/2 antibodies detect the constant region of the TCRβ chain, they cannot be used to assess clonality of γδ T cells, which do not express TCRβ. Methods for assessing clonality of γδ T cells by flow cytometry have not been validated. Second, because surface expression of the TCR complex requires surface CD3 expression, T-cell populations that have lost surface CD3 cannot be assessed for clonality by surface TRBC staining because such populations will be negative for both surface TRBC1 and TRBC2. Molecular techniques: TCR gene rearrangement may be assessed at the molecular level by polymerase chain reaction–based methods or, more recently, by NGS. These studies report clonal populations as fractions of total sequencing reads, corresponding to fractions of the total rearranged T-cell repertoire. The threshold for clonality may vary among platforms and laboratories and is inherently arbitrary. One important caveat of TCR rearrangement studies is that it is not definitively possible to infer whether a T-cell clone corresponds to an αβ TCR clone or a γδ T-cell clone because rearrangement of the β, γ, and δ loci occurs approximately concurrently.⁷⁴ In addition, it is not possible to definitively correlate a particular clonal sequence identified by polymerase chain reaction– or NGS-based methods with a clonal population identified by flow cytometry. Appropriate interpretation of T-cell clonality requires holistic integration of both the relative and absolute abundance of the clone, together with immunophenotypic and clinical data.

Although these methods are invaluable for the diagnosis and monitoring of mature T-cell neoplasms, their increased sensitivity has also led to the frequent identification of T-cell clones in patients without clinicopathologic features of T-cell malignancy. Such T-cell clones, neither meeting diagnostic criteria for a T-cell neoplasm nor occurring in patients with clinical suspicion of a T-cell neoplasm, are detected in up to 26% of patients undergoing workup for hematolymphoid neoplasm and 13% of healthy donors.1, 2, 3 Because these clones do not meet diagnostic criteria for a T-cell neoplasm, they are commonly labeled T-cell clones of uncertain significance (TCUS). However, this entity remains poorly understood, and consensus guidelines for reporting, surveillance, and management have not yet been proposed. As such, the detection and reporting of such unexpected T-cell populations pose risk of unnecessary diagnostic procedures, can generate unwarranted patient anxiety, and, in rare cases, even lead to inappropriate therapeutic interventions.

TCUS has been compared to other clonal lymphoid populations of uncertain significance, such as monoclonal B-cell lymphocytosis and monoclonal gammopathy of uncertain significance.4, 5, 6, 7 It has therefore been argued that TCUS may represent premalignant or incipient T-cell lymphomas, particularly T-large granular lymphocytic leukemia (T-LGLL), which shares significant immunophenotypic overlap with many TCUS.¹ Thus, it has been proposed that any persistent T-cell clone is abnormal and should be considered premalignant.^4,6

However, to our knowledge, there have been no longitudinal studies demonstrating the evolution of TCUS to T-LGLL. Additionally, the prevalence of TCUS is believed to exceed 10% in healthy individuals,¹ and given that the incidence of T-LGLL is <1 per million person-years,⁸ this implies a theoretical evolution rate of <1 per 100 000 person-years. Such an evolution rate of TCUS to T-LGLL is far too low to warrant close surveillance of every TCUS. We therefore argue that most TCUS have limited premalignant potential and instead contend that there is abundant evidence indicating that persistent clonal T-cell populations are frequently generated by nonneoplastic mechanisms (Figure 1). Given the discrepancy between the prevalence of TCUS and the published incidence of T-LGLL, guidance on reporting, surveilling, and managing such unexpected clones is needed. To encompass each of the possible pathophysiologic mechanisms that may underlie an incidental T-cell clone, for the purpose of this article, we define a TCUS as any T-cell clonality detectable by flow cytometry or molecular techniques that does not meet diagnostic criteria for a T-cell neoplasm. We discuss the various mechanisms that may generate a detectable T-cell clone and provide a risk-stratification strategy to help pathologists and clinicians recognize, report, and manage common clinicopathologic patterns of incidental T-cell clonality.

Immunologic mechanisms of nonneoplastic T-cell clonality

T-cell clones may be generated by several nonneoplastic mechanisms. When this occurs in response to a particular antigen-specific activation, we refer to these clones as reactive. Conceptually, any process that reduces the T-cell repertoire diversity increases the likelihood of identifying a clone. In the absence of T-cell neoplasia, T-cell repertoire diversity is most often reduced by 1 of 2 mechanisms: (1) immune activation leading to proliferation of antigen-specific T-cell clones; or (2) a decrease in the total size of the T-cell repertoire (eg, lymphopenia; Figure 1).

An important consequence of T-cell activation is clonal proliferation, ensuring that sufficient antigen-specific T cells are available to mount an effective immune response. Thus, any sufficiently robust immune activation can result in a clonal expansion detectable by molecular techniques or flow cytometry. For instance, in acute infectious mononucleosis, the Epstein-Barr virus (EBV)–specific cytotoxic T-cell response is so profound that individual EBV-specific CD8⁺ T-cell clones can dominate >30% of the TCR repertoire.⁹ Similar magnitude clonal expansions also occur in human cytomegalovirus (CMV) infection, in both the CD8⁺ and CD4⁺ T-cell compartments.^10,11 Because next-generation sequencing (NGS)–based TCR clonality assays commonly set clonality thresholds between 2.5% and 5%,^12,13 clones of such magnitude are readily detectable in clinical laboratory testing.

The final phase of T-cell response to a pathogen is contraction, in which many clonal effector T cells undergo apoptosis to maintain immune homeostasis.¹⁴ Thus, some groups have proposed that persistence of a T-cell clone excludes a reactive or nonneoplastic etiology and is expected to carry some degree of inherent malignant potential.⁴ Unfortunately, the kinetics of the contraction phase are highly variable and dependent on the persistence of the stimulating antigen, and many virus-specific T-cell clones may persist even in the absence of ongoing infection or detectable viral reactivation.^15,16 This is particularly evident for human herpesvirus infections such as EBV and CMV, which are controlled by lifelong effector T-cell responses and may result in long-term maintenance of virus-specific clones influenced by the immune status of the host.17, 18, 19 In addition, extensive rounds of proliferation in response to persistent antigen stimulation will result in replicative senescence.²⁰ Data from animal and cell culture models suggest that although senescent T-cell clones do not have proliferative capacity, they are resistant to apoptosis, resulting in their persistent detection through aging.^21,22 Nonneoplastic T-cell clonality is thus more common in older individuals and in patients with underlying autoimmunity or chronic infection (Figure 1), because these are settings characterized by repeated immune activation, cumulative T-cell repertoire narrowing, and eventual immunosenescence.23, 24, 25, 26 Therefore, persistence of a clonal T-cell population alone is insufficient to ascribe malignant potential to that clone.

T-cell repertoire diversity also decreases in the setting of lymphopenia, which reduces the overall size of the T-cell repertoire. Lymphopenia is common among patients receiving chemotherapy and those who have received hematopoietic stem cell or solid organ transplants. In addition to decreasing T-cell repertoire size, lymphopenia also induces compensatory clonal T-cell expansions via homeostatic proliferation, in which residual T cells proliferate to maintain the total size of the T-cell pool at a near-constant level.27, 28, 29, 30, 31, 32 Homeostatically expanded clones can reach levels detectable by clonality assays, mimicking neoplastic proliferations by molecular or flow cytometric criteria.

Importantly, T-cell clones generated through the mechanisms described above do not inherently have malignant potential and therefore should not be considered premalignant. This distinguishes some TCUS from premalignant hematolymphoid states such as monoclonal B-cell lymphocytosis, monoclonal gammopathy of uncertain significance, and clonal hematopoiesis of indeterminate potential, each of which has a demonstrable risk of malignant transformation and therefore merits reporting and clinical follow-up.33, 34, 35 Below, we discuss how to identify these reactive patterns to better determine the appropriate level of reporting, workup, and follow-up.

Toeing the line, TCUS and T-LGLL

The T-cell subset that is most likely to proliferate in reactive settings is highly antigen-experienced effector memory T cells, especially those of cytotoxic phenotype. Thus, the most common diagnostic dilemma is distinguishing an incidental T-cell clone with a cytotoxic phenotype from T-LGLL, a neoplasm most often arising from T cells with a cytotoxic and effector memory phenotype.^4,36,37 This distinction is particularly relevant for CD8⁺ clonal populations, because symptomatic T-LGLL is typically CD8⁺, whereas CD4⁺ T-LGLL is generally indolent and rarely require treatment.³⁸ Distinguishing reactive large granular lymphocyte (LGL) expansions from T-LGLL is challenging for several reasons. First, T-LGLL are often identified incidentally and may present only with nonspecific clinical features such as unexplained cytopenias. Although activating mutations in the JAK/STAT pathway, most commonly in STAT3 and STAT5B, are key pathogenic drivers of T-LGLL, they are detected in only ∼50% of patients; hence, their absence does not exclude the diagnosis.39, 40, 41 Furthermore, T-LGLL is strongly associated with nonhematologic conditions that may also drive reactive cytotoxic T-cell expansions, such as rheumatoid arthritis and solid tumors.42, 43, 44, 45, 46, 47

It remains controversial whether reactive LGL expansions in patients with these nonhematologic conditions represent true hematolymphoid malignancy or exaggerated reactive responses to antigenic stimulation. Limited reports have documented the disappearance of LGL clones after resolution of the primary nonhematologic disease, suggesting the benign nature of these clones.^36,48,49 Additionally, transient LGL expansions after hematopoietic stem cell transplant may predict favorable outcomes, possibly reflective of a graft-versus-leukemia effect.50, 51, 52 Conversely, there is recent evidence that significant antigenic pressure may rarely lead to LGLL neoplasia (Figure 1). For instance, inborn errors of immunity may underlie T-LGLL pathogenesis, leading to persistent LGL proliferations that can acquire Jak/Stat mutations.⁵³ Furthermore, there is an overlap in leukemic vs nonleukemic TCR repertoires in patients with STAT-mutated LGLL, suggesting persistent antigenic stimulation as a driver.^40,54

Unfortunately, because nonneoplastic T-cell clonality in healthy individuals frequently shares immunophenotypic features with T-LGLL, distinguishing between these conditions remains a major diagnostic challenge. A seminal study by Shi et al demonstrated that there was no immunophenotypic feature capable of distinguishing TCUS from LGLL.¹ As such, the authors proposed an absolute count threshold of <0.5 × 10³/μL or <20% of lymphocytes in peripheral blood, below which the clone is most likely to be nonneoplastic and not require further diagnostic workup. This threshold provided superior differentiation between nonneoplastic vs neoplastic T-cell clonality with LGL phenotype compared to relying on immunophenotypic features. Notably, this threshold was suggested for peripheral blood specimens, and no such threshold has been established for bone marrow or tissue specimens. Although there is no primary evidence evaluating this threshold outside of T-LGLL, we argue it represents a sensible starting point for assessing a T-cell clone in the absence of clinical suspicion for T-cell neoplasm.

Clinicopathologic features of benign and neoplastic T-cell clonality

Features that increase suspicion for a malignant process include the following: (1) large clones that increase in abundance over time; (2) clones associated with clinical or morphologic features suggestive of T-cell neoplasms; and (3) clones exhibiting distinct immunophenotypic abnormalities more specific to T-cell neoplasms, such as complete loss of surface CD3 or CD2, or aberrant expression of CD10 or CD30.⁵⁵ Loss of surface CD3 expression is a feature more typical of angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, lymphocyte-variant hypereosinophilic syndrome, hepatosplenic T-cell lymphoma, and T-acute lymphoblastic leukemia, which generally comprise more aggressive entities.56, 57, 58, 59, 60 Identification of a discrete CD3⁻ T-cell population should prompt comprehensive diagnostic evaluation. Additionally, although the frequency of LGL-phenotype CD4⁺ T-cell clones may vary between laboratories depending on panel design and analysis strategies, non–LGL-phenotype CD4⁺CD8⁻ T-cell clones may be regarded as suspicious.¹ Importantly, partial loss of canonical T-cell antigens CD5 or CD7 is common in reactive LGLs and should not be considered definitive evidence of malignancy.^1,55 Similarly, expression of so-called “natural killer (NK) antigens,” such as CD16, CD56, CD94, NKG2 receptors, or killer cell immunoglobulin-like receptors (KIR), is frequent in reactive cytotoxic T cells and not inherently aberrant.^55,61,62 Paradoxically, the World Health Organization fifth edition includes several of these features as diagnostic criteria for T-LGLL,⁸ underscoring the diagnostic ambiguity in this area.

Our approach to evaluating T-cell clonality of uncertain significance

We present our recommendations for reporting TCUS identified in peripheral blood, along with additional diagnostic workup and clinical follow-up, in Figure 2. Incidental T-cell clones may originally be identified by flow cytometry or molecular techniques including polymerase chain reaction or NGS. Any T-cell clone identified by molecular techniques should undergo immunophenotypic characterization by flow cytometry (Figure 2, Box 2). αβ T-cell clones that retain surface CD3 expression should be quantified. We propose that the absolute count threshold established for T-LGLL¹ (<0.5 × 10³/μL or <20% of lymphocytes) can be applied to CD3⁺ T-cell clones in patients without clinical or laboratory evidence of T-cell neoplasia. In these cases, we recommend against reporting such clones in the top line of diagnostic reports, because they are likely not malignant or premalignant and their reporting often triggers unnecessary invasive procedures (eg, bone marrow biopsy).

Figure 2.

Algorithm for approaching peripheral blood T-cell clonality in patients without established diagnosis of T-cell neoplasm. ∗Clinical features of T-cell neoplasm: hepatosplenomegaly, lymphadenopathy, diffuse rash, B-symptoms, unexplained persistent cytopenias, unexplained eosinophilia, and autoimmune hemolysis. †Large granular lymphocyte phenotype: CD57⁺, CD5 dim, CD7 dim to negative, CD2 normal to bright, and CD16 and CD56 negative or dim variable. §T-cell clone reporting recommendations for pathologists. TCUS: do not report a TCUS in the top line of a diagnostic report, because this will frequently prompt unnecessary diagnostic workup. A population meeting criteria for TCUS as defined in this algorithm should be documented in a comment or other interpretation section for surveillance purposes. If multiple TCUS are identified in a specimen, we recommend reporting that "multiple small TRBC-monotypic T-cell populations are identified, compatible with reactive oligoclonal expansion." If this is observed in the setting of lymphopenia, we recommend commenting that this finding is consistent with homeostatic proliferation. Low risk: for populations meeting criteria for "low risk" in this algorithm, we recommend against reporting this in the top line of a diagnostic report. Instead, we recommend reporting the clone in a comment, documenting its absolute and relative abundance, and recommending clinical and laboratory surveillance. Abnormal population: report in the top line of diagnostic report, document immunophenotype as well as absolute and relative abundance. ‡Workup recommendations for clinicians. Low risk: for clones meeting the "low-risk" criteria defined in this algorithm, we recommend surveillance with repeat CBC and clinical follow-up at an interval of 3 to 6 months. We also typically perform repeat flow cytometry at this time to access for resolution, stability, or progression of the clone. Abnormal population: upon flow cytometric characterization of an abnormal circulating T-cell population by this algorithm, we recommend beginning workup with molecular testing on peripheral blood. For clones suspicious for T-LGLL, consider STAT3 and STAT5B molecular testing for CD8⁺ and CD4⁺ clones, respectively. Imaging studies for staging may also be considered when indicated. ∗∗Note: Distinguishing between CD4⁺ TCUS and CD4⁺ LGLL is not critical, because CD4⁺ LGLL rarely requires treatment, even when pathogenic STAT5B mutations are present.

Box 2. Clinical and morphologic correlation for abnormal CD4/CD8 double-positive or double-negative populations.

Double-positive: CD4/CD8 double positivity is a frequent immunophenotype of benign TCUS. These populations generally show tight coexpression of CD4 and CD8 and are often either CD4^dimCD8⁺ or CD4⁺CD8^dim. These populations generally account for <20% of lymphocytes and <0.5 × 10³/μL, are frequent in older patients, and, in the absence of clinical suspicion for T-cell neoplasm, may be safely reported in a comment or other interpretation section. Double-positive T-cell clones more abundant than these thresholds, or occurring in a patient with a high clinical suspicion for lymphoma or leukemia, should prompt consideration for a T-cell neoplasm. Although any T-cell neoplasm may present with a double-positive immunophenotype, the most common entities are T-prolymphocytic leukemia and T-lymphoblastic leukemia, both of which have characteristic morphologies that can help inform the differential diagnosis.⁵⁵ Double-negative: double-negative (CD4⁻CD8⁻) clones are less common; care should be exercised to confirm that a double-negative population does not represent γδ T cells, which by definition do not express TRBC1/2. A double-negative αβ T-cell clone should raise suspicion for autoimmune lymphoproliferative disorders such as autoimmune lymphoproliferative syndrome or Ras-associated lymphoproliferative disease in the appropriate clinical setting. Although expansions in these conditions are typically polyclonal, some may demonstrate restricted T-cell repertoires.⁷⁵

For clones exceeding this threshold or those identified in patients with clinical suspicion of T-cell neoplasm, additional workup is warranted. LGL-phenotype clones with an absolute count <2000 cells per μL should be considered low risk in the absence of persistent unexplained cytopenia (particularly neutropenia), meriting surveillance. Unless flow cytometry or clinical findings strongly suggest overt or aggressive T-cell neoplasm, we recommend initial molecular testing on peripheral blood rather than immediate bone marrow biopsy. If recurrent molecular abnormalities associated with T-cell neoplasia are identified, bone marrow biopsy and appropriate imaging studies can then be pursued for staging and complete diagnostic workup.

Case 1

A patient in their 30s with no significant past medical history was noted to have persistent thrombocytopenia (108 × 10³/μL). The patient had no other cytopenias except mild lymphopenia and was asymptomatic. Peripheral blood flow cytometry reported the presence of 2 abnormal T-cell populations, both meeting the accepted criterion for TRBC restriction, that is, ≥85% of a CD3⁺TCRγδ⁻ population being either TRBC1⁺ or TRBC1⁻.⁵⁵ This report prompted referral to hematology for workup of a possible T-cell lymphoma with anticipated bone marrow biopsy.

On evaluation by hematology at our institution, the patient remained asymptomatic and had similar complete blood count (CBC) findings. Repeat peripheral blood flow cytometry was ordered, as well as TCR rearrangement studies by NGS and molecular testing by a panel of 203 mutations recurrent in hematolymphoid malignancy (Stanford Actionable Mutation Panel for Hematopoietic and Lymphoid Malignancies; Heme-STAMP; Figure 3A-B).

Figure 3.

Three cases of incidental T-cell clones. (A-B) Case 1: clinical history and pertinent laboratory results (A) and flow cytometry immunophenotyping (B). (C-D) Case 2: clinical history and pertinent laboratory results (C) and flow cytometry results showing restricted T cells (orange) and NK cells (blue) (D). (E-H) Case 3: clinical history and pertinent laboratory results (E) and flow cytometry immunophenotyping (F). (G) Longitudinal analysis of clonal TCR sequences present at diagnosis and present in the current specimen. (H) Immunophenotypic aberrancies at diagnosis compared to immunophenotype of atypical T-cell population in current specimen. ALC, absolute lymphocyte count; ANC, absolute neutrophil count; FITC, fluorescein isothiocyanate; Hgb, hemoglobin; PD-1, Programmed cell death protein 1; PE, phycoerythrin; WBC, white blood cells.

Flow cytometry demonstrated 2 populations meeting criteria for TRBC restriction: 1 CD4⁺ population constituting 12.2% of lymphocytes (0.1 × 10³/μL; 96% TRBC1⁺) and 1 CD8⁺ population constituting 7.5% of lymphocytes (0.06 × 10³/μL; 85% TRBC1⁻). The CD4⁺ population expressed CD57 and demonstrated dim-to-negative CD7, dim CD4, and slightly bright CD2, all features compatible with LGL immunophenotype (Figure 2). The CD8⁺ population was immunophenotypically heterogeneous and also displayed features compatible with LGL immunophenotype. TCR gene rearrangement studies were clonal for TCRβ rearrangement (the most abundant clone making up 5.7% of reads); TCRγ was oligoclonal. Molecular studies on peripheral blood were negative for abnormalities recurrent in hematolymphoid neoplasms.

Discussion on case 1

In this case, an asymptomatic patient with low clinical suspicion for T-cell lymphoma was found to have 2 T-cell clones by flow cytometry, each accounting for <20% of lymphocytes and each less abundant than 0.5 × 10³/μL. By the criteria proposed in Figure 2, these populations are compatible with TCUS and do not require top line reporting or further workup.

This case illustrates a common pattern of oligoclonal T-cell expansions, likely due to homeostatic proliferation in the setting of lymphopenia. Flow cytometry shows 2 T-cell populations meeting criteria for restriction, both displaying immunophenotypic features of effector memory T cells. The CD8⁺ population barely crosses the threshold for restriction (85%) and appears immunophenotypically heterogeneous, and TCRβ rearrangement studies barely cross our assay’s threshold for clonality (5%). The immunophenotype of the CD4⁺ clone is extremely common among patients with lymphopenia with oligoclonal expansions in our clinical practice. Additionally, isolated thrombocytopenia is uncommon in T-LGLL. Negative molecular studies provide further reassurance against underlying hematolymphoid malignancy.

Final diagnosis

T-cells without immunophenotypic abnormality.

Recommended clinical action

Routine clinical follow-up is recommended. The patient’s mild thrombocytopenia may represent immune thrombocytopenia, which does not require intervention without symptoms and with a platelet count >100 × 10³/μL.

Case 2

A patient aged <10 years, receiving maintenance chemotherapy for B-lymphoblastic leukemia, was noted to have a CD8⁺ T-cell clone on flow cytometry performed for disease monitoring. The patient’s CBC showed lymphopenia (0.87 × 10³/μL) and thrombocytosis (462 × 10³/μL), and additional laboratory testing demonstrated active CMV viremia (Figure 3C). The patient was asymptomatic, except for nausea after chemotherapy sessions, and otherwise had no active clinical concerns. TCR gene rearrangement studies were added to assess the T-cell clone reported by flow cytometry, which returned positive for TCRβ and TCRγ clonality, with the most abundant clone constituting 30% of reads. The patient was scheduled for a bone marrow biopsy to assess the significance of the T-cell clonality; before undergoing the procedure, repeat peripheral blood flow cytometry study was performed, as shown in Figure 3D.

Flow cytometry redemonstrated a TRBC1-restricted CD8⁺ T-cell population accounting for 27.7% of lymphocytes and estimated at 0.24 × 10³/μL. The population was negative for CD5, demonstrated dim CD7, and was uniformly positive for CD56 and CD57. Additional analysis of NK cell receptor expression showed that the T-cell clone was uniformly positive for an individual KIR (CD158b), as well as NKG2C, an NK cell receptor involved in recognizing CMV-infected cells. Additionally, an NK cell clone was identified, which uniformly expressed an individual KIR (CD158a) and NKG2C.

Discussion on case 2

Here, a patient with low clinical suspicion for a T-cell neoplasm was found to have a T-cell clone <0.5 × 10³/μL but accounting for >20% of lymphocytes. The T-cell clone was CD8⁺ and displayed immunophenotypic features of activated LGLs. We consider this clonal population low risk and recommend surveillance with repeat CBC (Figure 2).

This case illustrates the magnitude of reactive clonal expansions that may occur in response to viral infection/reactivation. This population should be considered benign because it is low in absolute abundance and identified in a patient with low suspicion for a T-cell neoplasm. Furthermore, the patient had active CMV viremia, and the atypical population expressed NKG2C, a receptor involved in detecting CMV-infected cells.63, 64, 65 A concurrent NKG2C⁺ NK cell clone was also identified, further suggesting the reactive nature of these populations. Interestingly, the atypical population demonstrated striking immunophenotypic abnormalities, including complete loss of CD5 and uniform expression of CD56; these features are classically described as characteristic of T-LGLL and highlight the low specificity of these immunophenotypic findings in distinguishing T-LGLL from benign T-cell clones.

On routine clinical follow-up 2 months later, the patient remained asymptomatic, had no significant changes in CBC indices, and repeat flow cytometry demonstrated that both clonal T-cell and NK cell populations had slightly decreased compared to before. These features are collectively reassuring and should prompt clinical and laboratory surveillance rather than invasive procedures for further workup.

Final diagnosis

T-cells without diagnostic abnormality (see “Diagnosis comment”).

Diagnosis comment

There is no morphologic or immunophenotypic evidence of hematolymphoid neoplasm.

We note the patient's CMV viremia and lymphopenia in the setting of ongoing chemotherapy. A clonal TRBC1^dim CD8⁺ T-cell population is redemonstrated at an estimated abundance of 0.24 × 10³/μL. The clonal T-cell population expresses NKG2C, and there is also a CD158a⁺b⁻e⁻-restricted NK cell clone expressing NKG2C (0.05 × 10³/μL). NKG2C is an NK cell receptor involved in recognizing CMV-infected cells. Given the low absolute counts of these populations and immunophenotypes associated with CMV viremia, these populations are strongly favored to represent reactive clonal expansions.

Recommended clinical action

Routine clinical follow-up and surveillance with repeat CBC are recommended.

Case 3

A patient in their 80s with a 12-year history of Sézary syndrome presents for follow-up. The patient had undergone multiple lines of therapy before ultimately achieving complete response with pembrolizumab 3 years ago. Since then, the patient has remained asymptomatic and has not been receiving therapy for their T-cell lymphoma.

Approximately 1 year ago, the patient underwent repeat peripheral blood immunophenotyping for disease monitoring, which showed an “atypical T-cell population concerning for residual/recurrent disease.” TCR gene rearrangement studies were positive for clonal rearrangements of TCRβ and TCRγ. Before anticipated reinitiation of treatment, the patient underwent repeat peripheral blood flow cytometry (Figure 3E-H).

Flow cytometry demonstrated a TRBC1⁺ T-cell population making up 7.5% of lymphocytes (0.11 × 10³/μL). The population was double positive for CD4 and CD8 and showed aberrant loss of CD7 and CD26 (Figure 3F).

Discussion on case 3

In patients with an established diagnosis of T-cell lymphoma, it is important to compare immunophenotyping and clonality results to prior studies when available. Review of prior results for this patient showed that the immunophenotype of the neoplastic population at diagnosis and through 9 years of treatment was distinct from the atypical population in this study (Figure 3H). Although the immunophenotype of a neoplastic population can change over time, particularly in the posttherapy setting, several features favor that the current atypical population is unrelated to the patient’s T-cell lymphoma. First, coexpression of CD4 and partial CD8 is uncommon for Sézary syndrome, and acquisition of CD8 by a CD4⁺ mycosis fungoides is rare and associated with aggressive clinical features,⁶⁶ incompatible with this case. Instead, the phenotype of this clone is more compatible with a CD4⁺CD8⁺ double-positive TCUS that is frequently identified in older patients. More definitively, however, review of TCR sequencing results indicate that the clonal sequences in the current specimen do not match those present in the diagnostic specimen, suggesting the clone is unrelated to the patient’s T-cell lymphoma (Figure 3G).

Given that the clonal population identified in this study is unlikely to represent residual/recurrent disease, we can treat this population according to the criteria presented in Figure 2. The clone is <20% of lymphocytes, less abundant than 0.5 × 10³/μL, and identified in a patient with low suspicion for an active T-cell neoplasm; therefore, this clone is likely benign and should not prompt additional workup or treatment. Because TCUS is commonly identified in patients with a history of mycosis fungoides/Sézary syndrome,⁶⁷ this case highlights that caution should be exercised before changing management when new dominant clonal sequences or major immunophenotypic shifts are identified in asymptomatic patients with a history of T-cell lymphoma.

Final diagnosis

T-cells without diagnostic abnormality (see “Diagnosis comment”).

Diagnosis comment

Flow cytometry demonstrates an atypical T-cell population immunophenotypically dissimilar to the patient’s previously reported T-cell lymphoma. We note that concurrent NGS studies reveal a clone that was not the dominant clone in the diagnostic specimen. Although we cannot definitively exclude that this population represents a subclone of the T-cell neoplasm, we favor that the population represents an unrelated reactive clonal expansion.

Recommended clinical action

Routine clinical follow-up is recommended.

Conclusions

The increasing use of sensitive T-cell clonality assays, including TCR rearrangement studies by NGS and flow cytometry staining for TRBC1/2, have improved our ability to detect T-cell neoplasms while simultaneously uncovering a broad landscape of benign T-cell clones in healthy individuals. These clones, commonly generated by infection or homeostatic proliferation, generally have no malignant or premalignant potential. Appropriate diagnosis of these entities requires careful synthesis of immunophenotypic features, clone size, and clinical context.

Substantial work, particularly longitudinal studies incorporating molecular studies and clinical outcomes, is needed to define evidence-based thresholds that reliably separate benign clonality from early T-cell neoplasia. Although we await such consensus criteria, the framework proposed here offers a practical and conservative approach to interpreting T-cell clonality that minimizes overdiagnosis and unnecessary intervention while maintaining appropriate sensitivity for true T-cell malignancy.

Conflict-of-interest disclosure: The authors declare no competing financial interests.