Bi‐Clonal ( <scp>CD4</scp> + and <scp>CD8</scp> +) T Cell Large Granular Lymphocytic Leukemia (T <scp>LGLL</scp> ) in an Asymptomatic Young Patient Confirmed by <scp>TRBC1</scp> on Flow Cytometry

Large granular lymphocyte (LGL) leukemia is a rare clonal disease characterized by the proliferation of large granular lymphocytes, leading to peripheral and marrow lymphocytic infiltration and cytopenia, mostly neutropenia. The major challenges lie in their distinction from reactive (including autoimmune) lymphoproliferations. Moreover, monoclonal LGL proliferative diseases are in fact a heterogeneous group of disorders, as recognized by the three subtypes in the current WHO classification [1]. LGLs are a distinct lymphoid subtype that are larger than typical circulating lymphocytes (15–18 μm) and have characteristic azurophilic granules containing acid hydrolases. LGLs constitute 10%–15% of normal peripheral blood mononuclear cells and arise from two major lineages. Approximately 85% are CD3 and CD57 positive and CD56 negative cytotoxic T cells, while the remaining 15% are CD3 negative and CD56 positive natural killer (NK) cells [2, 3]. Emerging evidence underscores the clinical and phenotypic importance of specific mutations in T-LGL leukemia, particularly activator of transcription 3 (STAT3), which is prevalent in CD8+ T-LGL and gamma/delta T-LGL leukemias and has been shown to correlate with neutropenia and poor overall survival. Conversely, the STAT5b mutation found in approximately 30% of CD4+ T-LGL cases lacks prognostic impact, while rare CD8+ T-LGLs have a poor prognosis [4, 5]. The re-classification of chronic lymphoproliferative disorders of NK cells as NK-LGL leukemia in WHO-HAEM5 [1] reflects recent evidence demonstrating monoclonal or oligoclonal expansion of NK cells with similarities to T-LGL leukemia. These insights provide a foundation for a deeper understanding of the genetic landscape in T-LGL and NK-cell leukemia [6-9]. Overall, the diagnostic pitfalls in LGLL range from recognition of the subtle T-cell or NK-cell proliferation, repeated establishment of their clonality, distinction of the LGLL clone from normal (T-) lymphocytes by a unique immunophenotype as well as detection and interpretation of molecular aberrancies. A 41-year male presented to our clinical with incidentally diagnosed leucocytosis (22.4 × 109/L), on a routine blood work. CBC done at our institute showed a Hb of 12.4 g/dL, TLC of 23.1 × 109/L, Platelet of 188 × 109/L, with 86% lymphocytes. Peripheral smear revealed lymphocytosis, with predominance of medium to large sized granular lymphocytes. There was no significant medical history, no skin lesion, no organomegaly or any other symptoms. There was no history of any underlying autoimmune condition. Flow cytometric evaluation was performed on the peripheral blood sample. The samples were processed using a bulk lysis–stain–wash protocol, and a 10-color, three-tube antibody panel was applied, which was as follows. Tube 1: FITC-Lambda, PE-CD200, PerCPCy5.5: CD5, PeCy7-CD19/CD4, APC-CD56/Kappa, APC-H7-CD45, APC-R700-CD3, BV421-CD20, BV510-CD8, BV605-CD10, Tube 2: FITC-Gamma Delta, PE-TRBC1, PerCPCy5.5: CD5, PeCy7-CD4, APC-CD56, APC-H7-CD45, APC-R700-CD3, BV421-CD57, BV510-CD8, BV605-CD7, Tube 3: FITC-CD2, PE-CD26, PerCPCy5.5: CD28, PeCy7-CD4, APC-H7-CD45, APC-R700-CD3, BV510-CD8, BV605-CD7. Data acquisition were carried out on the BD FACS Lyric platform using BD FACSuite Software and demonstrated approximately 76% CD3-positive T cells with bright CD3 expression, which were positive for CD2 and CD7, with a normal CD4 versus CD8 distribution (~1.1:1). Additional tubes showed the presence of a large proportion of cells expressing CD57. These cells also divided well into single positive CD4+ and CD8+ populations, with a ratio of ~1:1. Both these populations were clonally restricted toward the TRBC1 and expressed CD56, with the CD8+ population showing a downregulation of CD5. Summarily, two abnormal populations of LGLs were noted. One expressing CD3, CD7, CD5, CD4, CD57, CD56 and were clonal for TRBC1; while the other expressing CD3, CD7, CD5 (dim), CD8, CD57, CD56 and clonal for TRBC1. (Figure 1) Clonality assessment was done using sanger sequencing, targeting the T cell receptor gene Beta (TCRB), which also showed bi-clonal peaks. As there was no clinical feature demanding treatment, he was put under observation with regular follow-up for the last 2 years. He is undergoing regular CBC and biochemical analysis. The TLC is varying between 18 × 109/L and 24 × 109/L. He recently developed diabetes mellitus and current TLC being 19.8 × 109/L. T-LGL is uncommon neoplasms accounting for < 5% of lymphoproliferative disorders in general with a little higher prevalence in the Asian subcontinent [10]. An underlying chronic antigenic stimulation, aided by escape from Fas mediated apoptosis and activation of survival pathways, has been proposed as the pathogenic mechanism of [11]. About a third of patients present without any symptoms. The predominant presenting features include the presence of a cytopenia with moderate lymphocytosis. Only a fraction of patients presents with lymphocytic leukocytosis. There is an absence of lymphadenopathy or hepatomegaly. Splenomegaly is noted in about 15%–20% of patients. The case described in this study was an absolutely asymptomatic patient, without any features of autoimmunity, infections or organomegaly. In majority of cases, it is difficult to distinguish the T-LGL leukemia from activated cytotoxic lymphocytes observed in conditions such as infections and autoimmune diseases. Therefore, clonality assessment using molecular methods, or more recently using flow cytometric TRBC1/2 assessment is needed to confirm the clonality of T-LGL cells. T-LGL cells typically exhibit a cytotoxic phenotype and display CD3+, TCRαβ+, CD8+, CD16+, CD45RA+, and CD57+; and are CD4−, CD5(dim), CD27−, CD28− Immunophenotype [12]. Uncommon cases are positive for CD4+/TCRαβ+ and some TCRγδ+ cases. A true biclonal T LGL leukemia with one CD4+ and other CD8+ clonal population, supported by the TCR clonality assessment is extremely rare and has only rarely been reported [13]. The present is similar extremely rare case of young asymptomatic patient who harbored two clonal population confirmed with the help of both flow cytometric based TRBC1 clonality as well as sanger sequencing based TCR beta rearrangement analysis. Asymptomatic patients should be frequently followed up for symptoms and disease progression. A “watchful waiting” approach is mostly adapted. An absolute neutrophil count of less than 500/μL, recurrent infections, transfusion-dependent anemia, or autoimmune diseases such as rheumatoid arthritis needing medication are indications for treatment. Because leukemic LGLs are constitutively activated cytotoxic cells, immunosuppressive medication is the cornerstone of T-LGL leukemia treatment along with antibiotic support and erythropoietin [14]. Since there was objective indication for the treatment, the patient was kept under watchful observation with regular fine and is doing fine till date. To conclude, this case highlights a rare incidence of bi-clonal (CD4+ and CD8+) large granular lymphocytic leukaemia (LGLL) in a young asymptomatic patient, where the clonal proliferation was demonstrated on multicolour flow cytometry incorporating TRBC1 antibody. K.G. and K.R. prepared the planned, designed and drafted the manuscript, K.R. and R.G. were involved in making the diagnosis, M.K.S. was involved in sample processing and analysis, N.A. conducted the TCR Clonality assay. The authors have nothing to report. The authors confirm that artificial intelligence (AI)-assisted technology was not used in the writing or editing of this manuscript, and no images were manipulated using AI. Waived. Verbal and written consent is taken. The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.