T-large Granular Lymphocyte Leukemia Research Articles

T-Large Granular Lymphocytic Leukemia with Hepatosplenic T-Cell Lymphoma? A Rare Case of Simultaneous Neoplastic T-Cell Clones Highlighted by Flow Cytometry and Review of Literature

Lymphoproliferative diseases are a heterogeneous set of malignant clonal proliferations of lymphocytes. Despite well-established diagnostic criteria, the diagnosis remains difficult due to their variety in clinical presentation and immunophenotypic profile. Lymphoid T-cell disorders are less common than B-cell entities, and the lack of a clear immunophenotypic characteristic makes their identification hard. Flow cytometry turned out to be a useful tool in diagnosing T-cell disorders and to resolve complicated cases, especially if the number of analyzable neoplastic cells is small. We present a case of a 55-year-old man with simultaneous lymphoproliferative neoplastic T-cell clones, one αβ and the other γδ, identified and characterized by flow cytometry (FC), exploiting the variable expression intensity of specific markers. However, the patient’s rapid decline made it impossible to define a differential diagnosis in order to confirm the identity of the γδ clone, which remains uncertain. This case is added to the few other cases already documented in the literature, characterized by the co-existence of T-large granular lymphocytic leukemia (T-LGLL)-αβ and T-LGLL-γδ/Hepatosplenic T-cell lymphoma (HSTCL). Our case underlines the key role of sensitive diagnostic tools in the assessment of potential relationship between the diagnosis, prognosis, and treatment in the two pathologies.

TCL-154 STAT3 Mutations are Associated With Differential Treatment Responses to Methotrexate and Cyclophosphamide in T-Large Granular Lymphocyte Leukemia

TCL-154 STAT3 Mutations are Associated With Differential Treatment Responses to Methotrexate and Cyclophosphamide in T-Large Granular Lymphocyte Leukemia

POSTER: TCL-154 STAT3 Mutations are Associated With Differential Treatment Responses to Methotrexate and Cyclophosphamide in T-Large Granular Lymphocyte Leukemia

POSTER: TCL-154 STAT3 Mutations are Associated With Differential Treatment Responses to Methotrexate and Cyclophosphamide in T-Large Granular Lymphocyte Leukemia

Genomic landscape of T-large granular lymphocyte leukemia and chronic lymphoproliferative disorder of NK cells: a single institution experience

LGLL is a rare and chronic lymphoproliferative disorder including T-LGLL and CLPD-NK. Here, we investigated the genomic profiles of LGLL with a focus on STAT3 and STAT5B mutations in a cohort of 49 patients (41 T-LGLL, 8 CLPD-NK). Our study indicated that STAT3 was identified in 38.8% (19/49) of all patients, while STAT5B occurred in only 8.2% (4/49) of patients. We found that STAT3 mutations were associated with lower ANC in T-LGLL patients. The average number of pathogenic/likely pathogenic mutations in STAT3/STAT5B-mutated patients was significantly higher than that in WT patients (1.78 ± 1.17 vs 0.65 ± 1.36, p = 0.0032). Additionally, TET2-only mutated T-LGLL (n = 5) had a significant reduction in platelet values compared with the WT (n = 16) or STAT3-only mutated T-LGLL (n = 12) (p < 0.05). In conclusion, we compared the somatic mutational landscape between STAT3/STAT5B WT and mutated patients and correlate with their distinct clinical characteristics.

T-large granular lymphocytic leukemia.

T-cell large granular lymphocyte (T-LGL) leukemia is characterized by clonal expansion of cytotoxic T cells resulting in cytopenia. The proliferation of clonal LGLs is caused by prolonged antigenic stimulation, which leads to apoptotic dysregulation owing mainly to the constitutive activation of survival pathways, notably the JAK/STAT pathway. Understanding how leukemic T-LGL persists can aid in the development of future immunosuppressive therapies. In this review, we summarize the diagnosis and current standard of therapy for T-LGL leukemia, as well as recent advances in clinical trials.

Long-Term Adverse Events of Total Marrow Irradiation (TMI)-Based Conditioning Chemotherapy in Hematopoietic Stem Cell Transplant Recipients Treated on a Phase I/II Trial

Hematopoietic stem cell transplant (HSCT) remains a curative option for many hematologic malignancies. However, disease relapse is still one of the major causes of death after transplant. Increasing the intensity of conditioning regimens may elicit more antitumor activity but not without additional risk of transplant-related morbidity and mortality. The use of total body irradiation (TBI), while it can be effective, carries the risk of long-term toxicity to extramedullary organs, as well as secondary cancers. We have previously demonstrated that the combination of myeloablative chemotherapy with intensity-modulated radiation therapy to the bone marrow, or TMI, delivered with a linear accelerator, does not increase the toxicity of transplant in the first year of follow up (PMID 29065747; PMID 25234438). However, the effects of TMI in long term survivors after hematopoietic stem cell transplant (HSCT) has not yet been described. In this study, we retrospectively analyzed 24 consecutive patients who received HSCT with a TMI-based conditioning regimen between 2014 and 2021. The median age was 55.5 (range: 20-70) years. The median post-transplant follow up was 37.8 (range 12.0-89.7) months. Of 24 patients, 9 were diagnosed with multiple myeloma (MM) and received an autologous HSCT conditioned with high-dose Melphalan combined with either 3 (n=3), 6 (n=1) or 9 (n=5) Gy TMI. 15 (10 with acute myeloid leukemia, 3 with myelodysplastic syndrome and 2 with chronic myeloid leukemia) received an allogeneic HSCT from either a matched or 1-antigen mismatched related or unrelated donor, combining TMI 9 Gy with myeloablative fludarabine-IV busulfan. We then analyzed the incidence adverse events reported in published studies of TBI-based HSCT in our patient population (Table 1). In our long-term HSCT survivors receiving TMI-based conditioning, we observed xerostomia in 9 cases (37%, 3 of which had eating impairment), osteopenia or osteoporosis in 8 (33%, 2 of which also had fractures), dental in 29% and cataracts in 29%. Three subjects developed chronic kidney disease (12.5%, 2 patients developed stage 3 and 1 developed stage 4), 2 developed hypothyroidism (8%) and 2 patients developed heart failure (8%, both with mild decrease in ejection fraction without clinical decompensation). Only one patient (4%) developed a secondary malignancy (T-large granular lymphocytic leukemia). One patient (4%) developed a chronic interstitial pneumonitis, which was of unclear etiology and resolved spontaneously, but severe enough to require oxygen. These rates were overall lower than those historically reported with TBI (PMID 29482775) and can also be attributed to the standard chemotherapy utilized in the conditioning regimens. In conclusion, TMI is a relatively safe treatment modality when included in myeloablative conditioning regimens for HSCT. The adverse events profile in our patient population was similar to that of TBI but with lower incidence rates for the majority of reported events. Phase 2 clinical trials using TMI in conditioning regimens are currently underway to assess HSCT outcomes and should guide its future use in HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pathogenesis and Treatment of T-Large Granular Lymphocytic Leukemia (T-LGLL) in the Setting of Rheumatic Disease.

A complex relationship exists between rheumatic diseases and cancer. This delicate balance between chronic inflammation and malignant cell transformation in hematologic neoplasms has been observed, but is not well defined. Large Granular Lymphocyte (LGL) leukemia is at the intersection of a clonal lymphoproliferative disease, chronic inflammation, and autoimmunity. The association between rheumatoid arthritis (RA) and the spectrum of Felty’s Syndrome is well-known. Other rheumatic disorders have been reported including systemic lupus erythematosus (SLE), Sjogren’s Syndrome (SS), vasculitis, Behcet’s Disease (BD) and systemic sclerosis. The association between T-LGLL and rheumatic disease pathogenesis has been hypothesized, but has not yet been fully understood. Components of a shared pathogenesis includes chronic antigen stimulation, JAK-STAT pathway activation and overlap of various cytokines. We will summarize current knowledge on the molecular understanding between T-LGLL and rheumatic disease. There are many potential areas of research to help meet this need and lead to development of targeted therapeutic options.

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαβ-Large Granular Lymphocytic Leukemia.

Simple SummaryTRBC1 expression analysis by flow cytometry (FCM) has been recently proved to be a useful, simple and fast approach to assessing Tαβ-cell clonality. The aim of this study was to validate the utility of this assay specifically for the diagnosis of T-cell clonality of T-large granular lymphocytic leukemias (T-LGLL), as more mature polyclonal Tαβ large granular lymphocytes (Tαβ-LGL) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. Our results showed that a TRBC1-FCM assay is also a fast and easy method for detecting T-cell clonality in T-LGLL based on altered (increased or decreased) percentages of TRBC1+ Tαβ cells of LGL expansions (i.e., with lymphocytosis) suspected of T-LGLL, whereas in the absence of lymphocytosis (or in TαβCD4-LGLL), the detection of increased absolute cell-counts of more precisely defined subpopulations of T-LGL expressing individual TCRVβ families is required.Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1− Tαβ-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1− Tαβ-LGL ranged between 0.36 and 571 cells/μL (3.2–91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/μL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVβ families, the CD28− effector-memory and terminal-effector polyclonal Tαβ cells ranged between 0 and 25 TRBC1+ or TRBC1− cells/μL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1− cells/μL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Genomic landscape of TCRαβ and TCRγδ T-large granular lymphocyte leukemia

AbstractLarge granular lymphocyte (LGL) leukemia comprises a group of rare lymphoproliferative disorders whose molecular landscape is incompletely defined. We leveraged paired whole-exome and transcriptome sequencing in the largest LGL leukemia cohort to date, which included 105 patients (93 T-cell receptor αβ [TCRαβ] T-LGL and 12 TCRγδ T-LGL). Seventy-six mutations were observed in 3 or more patients in the cohort, and out of those, STAT3, KMT2D, PIK3R1, TTN, EYS, and SULF1 mutations were shared between both subtypes. We identified ARHGAP25, ABCC9, PCDHA11, SULF1, SLC6A15, DDX59, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3 as recurrently mutated putative drivers using an unbiased driver analysis approach leveraging our whole-exome cohort. Hotspot mutations in STAT3, PIK3R1, and FAS were detected, whereas truncating mutations in epigenetic modifying enzymes such as KMT2D and TET2 were observed. Moreover, STAT3 mutations co-occurred with mutations in chromatin and epigenetic modifying genes, especially KMT2D and SETD1B (P < .01 and P < .05, respectively). STAT3 was mutated in 50.5% of the patients. Most common Y640F STAT3 mutation was associated with lower absolute neutrophil count values, and N647I mutation was associated with lower hemoglobin values. Somatic activating mutations (Q160P, D170Y, L287F) in the STAT3 coiled-coil domain were characterized. STAT3-mutant patients exhibited increased mutational burden and enrichment of a mutational signature associated with increased spontaneous deamination of 5-methylcytosine. Finally, gene expression analysis revealed enrichment of interferon-γ signaling and decreased phosphatidylinositol 3-kinase–Akt signaling for STAT3-mutant patients. These findings highlight the clinical and molecular heterogeneity of this rare disorder.

Coexistence of T-Large Granular Lymphocyte Leukemia and Peripheral T Cell Lymphoma-NOS with indolent behaviour

T-cell lymphomas and leukemias are highly heterogeneous groups of rare disorders. We report a case of a 68-year-old man patient who developed two different T-cell neoplasms (Large Granular Lymphocyte Leukemia [LGLL] in 2018 and Peripheral T-cell non-Hodgkin lymphoma not otherwise specified [PTCL-NOS] in 2019) with a previous diagnosis of B-cell marginal zone lymphoma in 2010, treated with two lines of chemo-immunotherapy. The coexistence of these different T-cell neoplasms is rarely reported in the literature. Moreover, it is usually described as an LGLL transformation into PTCL-NOS; differently from these examples, herein, the simultaneous conditions appear to be driven by different T-cell clones. Furthermore, the PTCL-NOS had quite unusual behavior, with good disease control without intensive treatment. Because of these features, it could belong to a subgroup of indolent PTCL-NOS, not yet described in the WHO classification of T-cell neoplasms, which could benefit from less aggressive treatment.

Cyclophosphamide Induces Durable Molecular and Clinical Responses in Patients with Relapsed T-Large Granular Lymphocytic Leukemia (T-LGLL)

T-cell large granular lymphocytic leukemia (T-LGLL) is a clonal proliferation of cytotoxic T-lymphocytes that can result in severe cytopenias. The management of T-LGLL is immune-suppressive therapy, with methotrexate (MTX), cyclophosphamide (Cy), and cyclosporine (CsA) serving as primary frontline agents. While MTX has been the main front line agent to treat T-LGLL, overall response rates (ORR) are less than 40%, with complete response (CR) rates of only 5%. Data from the ECOG5998 (E5998) prospective trial and French cohort studies suggest an improved response with Cy in the 2 nd line setting. Anecdotal evidence suggests that Cy may eradicate the T-LGLL clone, producing complete molecular remission (CMR), which has not been observed with MTX or CsA. The degree to which a CMR can be attained and the lengths of such remissions with Cy remains unknown, particularly in the relapsed setting. We evaluated patients treated with Cy, to assess the duration of response and degree of CMR.We retrospectively evaluated patients treated for T-LGLL with oral Cy. Diagnosis of was based on 2016 World Health Organization Criteria. Patients needed a CD3+ CD8+ population on flow cytometry ≥500 cells/mm 3 and a positive monoclonal T-cell receptor (TCR) by PCR or restriction of TCR-Vbeta on flow cytometry. TCR-Vbeta rearrangement was deemed positive if one or more clone was detected in ≥10% of events. Disease response was defined by the E5998 study criteria. CMR was defined as CR by E5998 criteria and clearance of the TCR PCR gene rearrangement or TCR-VBeta flow cytometry. Time to response (TTR) was measured as time from start of Cy until partial response (PR) or CR, with patients who failed to respond being censored at the end of Cy treatment. Leukemia-free survival (LFS) in patients responding to Cy was measured as time from start of Cy until progression. Patients without progression were censored at last follow up. TTR and LFS were compared across variables using Kaplan-Meier curves with median survival and 95% confidence intervals.A total of 25 patients, with a mean duration of Cy treatment of 8 months, and median follow up time of 19 months, were included in this analysis. Patients were started on 50 mg daily for 2 weeks and then increased to 100 mg if tolerated. Three patients (12%) were treated with Cy as 1 st line, 14 (56%) as 2 nd line, 5 (20%) as 3 rd line, and 3 (12%) as 4 th line. Of the 3 patients that received Cy as 1 st line, none had a response. All refractory patients received MTX prior to Cy. Of the 22 refractory patients, 14 (64%) had a response (6 CR, 8 PR), 7 patients had no response, and 1 could not be determined due to development of multiple myeloma. Of the 6 patients that attained a CR, 50% had a CMR. The median TTR (CR or PR) was 6 months (95% CI: 4-7) while median time to PR was 9 months and median time to CR was 7 months. The median time to CMR was 11 months. In patients that achieved a response, median follow up time was 27 months, with a median LFS of 24 months. Median LFS for those who attained a PR was 20 months, while the median LFS for those who attained a CR was not reached as none progressed (Figure). The median follow-up for patients with a CMR was 17 months with LFS having not been reached due to no progression. There was no significant impact of age, gender, or presence of rheumatoid arthritis (RA) on LFS. Males (48%) had a shorter TTR compared to females (52%) (5 vs 7 months; p=0.05). Patients with RA (28%) also trended towards a shorter TTR (p=0.07).Herein, we demonstrate that patients treated for relapsed T-LGLL with Cy can attain durable remissions, with a prolonged response. Of particular interest is that no patients who attained a CR have relapsed, showing that durable remission is achievable, while no patients that received Cy as 1 st line had a response. Further, in patients that achieved a CR, 50% achieved a CMR which has not been previously demonstrated in the relapsed setting. While limited in cohort size, and additional follow up needed, these data suggest that Cy can produce long term remissions in patients with relapsed T-LGLL and induce CMR. While we demonstrate that CMR is attainable in the relapsed setting, the impact of this on long term disease control compared to clinical CR is unclear. Therefore, we recommend that CMR be used as an endpoint in future studies, particularly prospective trials, to evaluate response to treatment. These data clearly demonstrate that Cy is effective in the setting of relapsed T-LGLL and can induce long term disease control. [Display omitted] DisclosuresBrammer: Seattle Genetics: Speakers Bureau; Kymera Therapeutics: Consultancy; Celgene: Research Funding.

Endleukemia Assay v1: Enabling NGS-Based Comprehensive Routine Molecular Profiling of Leukemias in Routine Clinical Care

Background: Molecular biomarkers provide diagnostic, prognostic, predictive and minimal residual disease (MRD) information and constitute an integral part of diagnostic workup of hematologic neoplasms. Since a variety of different hematologic disorders can often show non-specific clinical symptoms as well as laboratory findings, the initial diagnostic workup needs to be optimized for timely and efficient coverage of a broad range of differential diagnoses. We developed a customized 81-gene panel, EndLeukemia Assay v1, for comprehensive genetic characterization of disorders that include bone marrow (BM) and peripheral blood (PB). The biomarkers on the panel cover genetic alterations in myeloid neoplasms (acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPN), aplastic anemia (AA), acute lymphoblastic leukemia (ALL), hairy cell leukemia, T-large granular lymphocytic leukemia (T-LGL) and T-prolymphocytic leukemia (T-PLL).Materials and Methods: Genomic DNA was isolated from fresh BM aspirate/PB or formalin-fixed paraffin-embedded (FFPE) BM clot specimens. Either entire coding sequences or limited exons/hotspots for 81-gene target genes were captured from 250 ng DNA using the hybridization-based HaloPlexTM target enrichment system (Agilent Technologies, Santa Clara, CA). These genes include A NKRD26, ASXL1, ASXL2, BCOR, BCORL1, BRAF, BRINP3, CALR, CBL, CBLB, CBLC, CEBPA, CREBBP, CRLF2, CSF3R, CUX1, DDX41, DNMT3A, EED, ELANE, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GFI1, GNAS, HNRNPK, HRAS, IDH1, IDH2, IKZF1, IL2RG, IL7R, JAK1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, MAP2K1, MPL, NF1, NOTCH1, NPM1, NRAS, PAX5, PHF6, PIGA, PML, PRPF40B, PTEN, PTPN11, RAD21, RARA, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG1, STAG2, STAT3, STAT5A, STAT5B, SUZ12, TERC, TERT, TET2, TP53, U2AF1, U2AF2, WT1, and ZRSR2. The panel design incorporated molecular barcodes to improve accuracy and sensitivity. Bidirectional paired-end sequencing was performed using the Illumina Miseq platform (Illumina, San Diego, CA). Variant calling was performed using Agilent SureCall software v3.5 and an in-house developed bioinformatics pipeline. Incorporation of single nucleotide polymorphisms (SNP) allows detection of sample identification errors and improves patient safety.Results: During the validation studies, high concordance was observed for missense mutations, insertions/deletions (indels), and selected copy number aberrations (CNAs) when compared with orthogonal methods. Under standardized conditions, a minimum average coverage depth of 3000x is achieved across the entire target region. The analytical sensitivity of the assay was established at 5% using a per base coverage cut-off of 250x. Of note, >90% of the CEBPA coding sequence, which is challenging to sequence due to a high GC component, was covered at >250x coverage depth. Copy number alterations including KMT2A partial tandem duplications (KMT2A -PTD) and IKZF1 deletions were detected successfully. Initial validation of FFPE tissue shows 100% concordance for all mutation calls for matched fresh and FFPE sample pairs. The ability to provide critical diagnostic and prognostic information across mutation hotspots from FFPE tissue if a fresh PB/BM sample is not available improves patient care. The top 25 mutations in the first 500 patient samples across different clinical indications are shown in the figure below. In addition, efficient panel design allowed uniform testing of follow-up bone marrow samples for the detection of MRD and clonal evolution.Conclusion: EndLeukemia assay v1 allowed comprehensive molecular profiling of a variety of hematologic neoplasms in routine clinical testing. Routine molecular biomarker profiling can be facilitated by incorporating smart design features in clinical NGS panels. [Display omitted] DisclosuresKantarjian:Delta-Fly Pharma: Research Funding; Novartis: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding.

Identification of a miR-146b-Fas ligand axis in the development of neutropenia in T large granular lymphocyte leukemia.

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4− phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

Clonal T-Large Granular Lymphocyte Proliferations in Childhood: Friend or Foe?

Background: The pathophysiology of clonal T-large granular lymphocyte (T-LGL) proliferations is not well understood; the distinction between reactive and malignant entities is not very clear given the fact that acquired STAT3 mutations can be seen with clonal T-LGL proliferations in non-malignant Felty syndrome and with frequent use of immunosuppression for the treatment of T-LGL leukemia. Historically, determination of clonality has been often seen as an indicator of T-LGL leukemia. We reviewed our experience on clonal T-LGL proliferations in children.Material and Methods: T-LGLs were identified by CD3 and dim CD5 staining on flow cytometric analysis and observation of lymphocytes with large granular cytoplasm appearance in peripheral blood. Cases with peripheral blood lymphocyte T-LGL population greater than 10% were reviewed in patients whom had flow cytometry studies performed for different indications. Among them, ones with available T cell receptor (TCR) gene rearrangement studies were selected and 16 cases with clonal TCR rearrangement patterns were included in this study. Fifty-seven peripheral blood flow cytometric tests using 15 surface markers were analyzed in those 16 cases. Patients have been followed for up to 7 years.Results: Sixteen cases of clonal T-LGL proliferation associated with different diseases were included in this analysis. Three patients had chronic graft versus host disease, 2 Evans syndrome, and one case each with the following conditions: common variable immunodeficiency disorder, severe combined immunodeficiency who developed CMV infection following umbilical cord blood transplantation (UCBT) and later acute EBV infection, co-existing Langerhans cell histiocytosis and primary hemophagocytic lymphohistiocytosis, autoimmune hemolytic anemia, chronic idiopathic thrombocytopenic thrombocytopenia, Hodgkin lymphoma, X-linked lymphoproliferative disorder, Rosai Dorfman disease (RDD), acute EBV infection, acute parvovirus B19 infection and paroxysmal nocturnal hemoglobinuria with history of treated severe aplastic anemia. Pathophysiological processes involved in these cases included various combinations of inherited immune deficiency in 4, acquired immune deficiency in 5, lymphoproliferation in 6, alloimmune reaction in 3, autoimmune reaction in 5, infection in 4, inflammation in 3 and malignancy in 1. One patient had three different clonal populations at different times due to maternal engraftment, CMV infection following UCBT and during an acute EBV infection after immune suppression withdrawal, respectively. The patient with RDD developed clonal T-LGL expansion when his disease flared. In some cases, clonal T-LGL proliferations disappeared with the resolution of the underlying pathological process. However, the T-LGL clone persisted and was frequently associated with relative increase over time, if the disease process continued to be active and/or progressed, even despite use of therapeutic approaches often including immunosuppression. Four clonal T-LGL proliferations in 2 patients have resolved, remaining 10 were persistent and have not been reevaluated in 4 cases. None of the cases showed a purely monoclonal pattern. The size of the clonal T-LGL population was variable with a median value of 18.6%, as high as 58.7% of the lymphocyte population. T-LGL population has positively correlated with CD3, CD8, CD57 (p<0.001) and inversely with CD19 (p=0.001) expression. There was also an overall positive correlation between the percent of T-LGL cells and absolute T-LGL count (p<0.001).Discussion: Clonal T-LGL proliferations in childhood is a reactive process reflecting the activity of the underlying disease process, including immunodeficiency, autoimmunity, alloimmunity, infection, inflammation, lymphoproliferation and malignancy. Use of immunosuppression has been associated with relative increase in the clone size despite decrease in peripheral blood absolute lymphocyte count along with relative reduction in T and B lymphocyte compartments in patients with active disease. Presence of clonal T-LGL proliferations in a polyclonal background is not an indicator of malignant process. It is very possible that such proliferations are an effort of the immune system to control the most prominently involved immunogenic stressors. DisclosuresNo relevant conflicts of interest to declare.

Clinical research progress of T-large granular lymphocytic leukemia

T-large granular lymphocytic leukemia (T-LGLL) is a rare T lymphocyte proliferative disease, and its clinical manifestations are splenomegaly, neutropenia and autoimmune diseases such as rheumatoid arthritis (RA). The pathogenesis of T-LGLL is to activate survival signaling pathway and inhibit apoptotic signaling pathway to maintain long-term activation of large granular lymphocytes (LGL) by chronic antigenic stimulation. Traditional treatment methods of T-LGLL are mainly immune inhibitors such as methotrexate, cyclophosphamide and other drugs, but now there are some new drugs such as tofacitinib in clinical research. Now, the clinical research progress of T-LGLL is reviewed. Key words: Leukemia, large granular lymphocytic; Etiology; Immunosuppressive agents

STAT3 mutation impacts biological and clinical features of T-LGL leukemia.

STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features.Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8±) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France).Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8± T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia.

Metronomic regimen as an effective treatment for aggressive T-LGL leukemia with central nervous system infiltration: clinical experience and review of literature.

A 71-year-old man was diagnosed with T-Large granular lymphocytic (LGL) leukemia, which usually represents a relatively indolent clinical course. While the clinical manifestation of this patient we report herein was aggressive with lasting fever, splenomegaly and hemophagocytic lymphohistiocytosis (HLH). T-cell immunophenotype was CD3+CD4-CD8-CD5-CD7-TCRαβ+. After comprehensive evaluation, an adjusted chemotherapy regimen CEOP (cyclophosphamide, vincristine, etoposide, prednisone) with etoposide, a potential effective regimen for HLH was administrated to the patient. Although he received intensive regimen, the patient showed drug resistance and disease progression with central nervous system (CNS) involvement during treatment and showed only transiently response to intrathecal methotrexate, cytarabine and dexamethasone. Therefore, considering the refractory elderly patient with fragile physical condition, metronomic regimen T-PEPC (oral administration of thalidomide, prednisone, cyclophosphamide, etoposide and methylhydrazine) was recommended, which refers to the frequent even daily administration of cytotoxic drugs at comparatively low doses with minimal or prolonged drug-free breaks. The patient responded well to this treatment and remained symptom-free for 8-month follow-up. To our knowledge, this is the first case of reporting this unique immunophenotype of dual CD4-/CD8- with aggressive clinical course and CNS involvement that successfully treated with metronomic regimen, suggesting that low dose metronomic regimen could be a better option for elderly patient with aggressive T-LGL leukemia.

Next-Generation Sequencing Analysis of Clonal Hierarchy and Dynamics in T-Large Granular Lymphocyte Leukemia Suggests Emergence of STAT3 Clones within Pre-Existing Dominant T-Cell Repertoire Responses Otherwise Silenced in Normal Individuals

T-large granular lymphocyte leukemia (T-LGLL) is a clonal lymphoproliferative disorder of cytotoxic T-cells (CTL) that is associated with cytopenias, predominantly neutropenia and reticulocytopenic anemia. From a scientific point of view, T-LGLL provides a natural model to study the dynamics of CTL responses; the heterogeneity of the disorder allows for examining the diversity of CTL responses in both autoimmune disorders and putatively chronic reactive conditions. A proportion of patients may have an extreme reactive process that mimics an indolent neoplastic lymphoproliferation. NGS and deep T-cell repertoire (TCR) sequencing provide insight into the clonal dynamics at work in T-LGLL patients.A large proportion of T-LGLL patients present with a bona-fide low-grade leukemia; this notion is supported by the discovery of recurrent somatic STAT3 mutations in some patients. STAT3 clonal burden represents an excellent marker that can be serially monitored along with clinical milestones to ultimately gain a more comprehensive understanding of disease etiology and natural history.We collected a cohort of 183 LGLL patients and screened them via deep NGS for mutation status of STAT3. In 36% of patients, 4 distinct somatic mutations (Y640F, N647I, D661V, D661Y) were identified in the SH2 domain of STAT3. In patients with wildtype STAT3, no somatic mutation was implicated in clonal expansion except for a small minority with STAT5 mutations present.We performed a longitudinal analysis of 20 representative STAT3-mutated T-LGLL patients with up to 10-year follow-up and an average of 7 analyzed blood samples per case. All serial samples were deep-sequenced to detect and determine the VAF of the known STAT3 mutations. Overall, STAT3 mutation VAF had a significant, inverse relationship to both hemoglobin and absolute neutrophil count (ANC) (both p<=0.001). In 7/11 cases harboring the Y640F mutation, chemotherapy led to remission accompanied by a decrease in VAF; 3 were asymptomatic and received no treatment. In patients with D661V or D661Y, 6/9 achieved remission with treatment. Only 1/3 cases with N647I entered remission.This longitudinal cohort can be sub-categorized into distinct patterns of clonal dynamics: 1) emerging STAT3 mutation in 20% of patients with a decrease in ANC as VAF of STAT3 clones expand; 2) an opposite trend in 40% of patients where VAF decreased due to therapeutic manipulations; 3) stable VAF in 20% of patients with little change over time; 4) codominant or dominant/secondary STAT3 mutations with distinct subclonal burden in 20% of patients.We performed deep TCR NGS on a representative subset of 9 patients to explore how STAT3 mutations correlated with T-cell clonal expansions. The data were processed by an extensive bioanalytic pipeline to quantify the relative abundance of each CDR3 rearrangement within a patient's TCR. Our cohort had an average of over 53,000 CDR3 templates per sample and was compared with 587 healthy controls.Our results demonstrate multiple patterns of clonal dynamics over the course of T-LGLL. Within each case, the immunodominant clones in serial samples were identified and correlated with STAT3 VAF burden over time. When patients were in remission, both STAT3 VAF and clonality were typically low. Interestingly, functional remission occurred in 2 cases despite increases in both clonality and STAT3 VAF. In 5/9 cases, the T-LGLL process involved 1 STAT3 mutation and 1 corresponding pathogenic clonotype displaying similar dynamics over time. In patients with 2 mutations, multiple high-frequency clonotypes were observed. Most significantly, comparison of STAT3 VAF and the dominant clonotype(s) revealed that STAT3 mutation can arise within a pre-existing clonal expansion that may harbor 2 branching mutations in extreme cases.Identification of CDR3 rearrangement sequences allowed for analysis of the distribution of clonotypes among patients and controls. The pathogenic clonotypes found in T-LGLL patients were detected in a high proportion of controls but at extremely low frequencies. This suggests that these potentially autoimmune clones exist in normal individuals but are effectively suppressed. No pathogenic clonotypes were shared among disease patients. In sum, analysis of clonal dynamics suggests that STAT3 mutations can occur in the context of pre-existing oligoclonal responses and involve otherwise low-frequency clonal specificities. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Mustjoki:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding.

Oligoclonal Expansion of Effector Memory CD8+CD57+ T Cells May Sustain Bone Marrow Destruction in Aplastic Anemia

The character of oligoclonal expansion of CD8+CD28- lymphocytes in aplastic anemia (AA), described by Risitano et al. (Blood, 2002 and Lancet, 2004), strongly suggests an antigen driving mechanism of T cell activation leading to destruction of hematopoietic stem and progenitor cells. In this study, we focused on a subset of CD8+CD28- T lymphocytes termed effector memory cells because of their high antigen-affinity and ability to be rapid activated after antigen stimulation. We investigated the frequency and oligoclonal expansion of effector CD28-CD57+ memory cells in CD4+ and CD8+ T subsets and the T cell receptor (TCR) Vβ repertoire by flow cytometry, and next-generation sequencing (NGS). Peripheral blood mononuclear cells from 20 AA patients and 14 healthy controls were evaluated for Vβ usage by flow cytometry. At the time of sampling, none of the patients had yet received immunosuppressive therapy. CD28+CD57- and CD28-CD57+ cells in CD4+ and CD8+ populations were assessed using the IOTest Beta Mark and a LSRII Fortessa cytometer for acquisition. The mean + 3SD of each Vβ group in controls was used as a cut-off to determine Vβ skewing in patients. When the frequencies of CD28+ and CD57+ cells in CD4+ and CD8+ subsets were compared, no differences were found between patients and controls (p=0.6125). Polyclonal expansion was described in CD8+CD28+, CD4+CD28+ and CD4+CD57+ T cells in 60%, 45% and 70% of patients, respectively. Using the mean of CD8+CD57+ cell frequency in controls as a cut-off, 12 of 20 patients displayed expansion of effector memory CD8+ cells and 11 of them (55% of all patients) showed expansion in 1 to 6 Vβ families with 1 to 4 immunodominant clones, while only 4 of 14 (29%) healthy controls displayed expansion of two Vβ families in expanded CD8+CD57+ cells without polyclonal expansion of CD8+CD28+ cells. In four cases (20% of all cases) of the remaining 8 patients whose cells did not display expanded CD8+CD57+ cells, there was expansion of 1 to 3 Vβ families with 1 dominant clone. These findings suggest activation of the immune response, polyclonal expansion of effector CD28+ compartments, and oligoclonal activation of effector memory CD8+ cells, regardless of frequency in peripheral blood. The frequency of CD8+CD57+ cells modestly correlated with the mean Vβ expansion in each patient (r2=0.5831, p<0.001). Also, we analyzed VDJ combinations and complementary region 3 (CDR3) sequences in CD4+ and CD8+ cells from 8 AA patients with CD8+CD57+ expansion in the ILLumina Hiseq 2000 sequencer. For the CD4+ compartment, patients were similar to healthy control CD4+ cells: frequencies of the most abundant clones less than 6%, CDR3 size profiles with Gaussian distribution and low degree of diversity (Simpson’s indexes range 0-1: 0 means infinite diversity, and 1 means no diversity). For CD8+ cells, 6 of 8 patients displayed a "skyscraper" Vβ/Jβ plot due to the presence of 1 to 3 dominant clones, with frequency greater than 10%, predominant classes in CDR3 size profile, and Simpson’s index similar to the CD8+CD57+ subset (p=0.1914). Analyzing CDR3 amino acid sequences for homology, we found that all the dominant sequences from patients were present at very low frequency in a healthy donor CD8+ cell pool (range, 0.12% - 53.6% vs 0.01% - 1.64%, p=0.0037), but no matches were described for the healthy CD8+CD57+ CDR3 repertoire. Only three CDR3 sequences were shared between patients. One of these CDR3 sequences (CSARDPPVSGTRGTDTQYF) was present in all patients and controls at very low frequency (mean expression, 5.01%), and it was the dominant clone in one patient, carried by TRBV20-1/TRBJ2-3 rearrangement at a frequency of 53.6%. Shared sequences were not found in reported viral TCR repertoires, nor in T cells recognizing peptide of myelin basic protein in multiple sclerosis, or synovial T cells from rheumatoid arthritis patients, nor matches were identified in T-large granular lymphocyte leukemia, AA, cancer or melanoma-derived TCR repertoires. Our data support the hypothesis of autologous immune-mediated attack leading to bone marrow destruction, likely triggered by autoantigens. Our results highlight the role of effector memory CD8+CD57+ T cells in sustaining an aberrant immune response during active disease. CD8+CD57+ cell expansion mirrors Vβ oligoclonal expansion, and Vβ flow cytometry should be useful in diagnosis and periodic evaluation of AA patients. DisclosuresFernandez Ibanez:GSK/Novartis: Research Funding. Young:Novartis: Research Funding.

[Analysis of Clinical Characteristics in 10 Patietns with T Large Granular Lymphocytic Leukemia].

To analyze the clinical manifestations and laboratory features of patients with T large granular lymphocytic leukemia (T-LGLL), so as to improve the understanding of this disease. The clinical data of 10 patients with T-LGLL in General Hospital of Chinese PLA from October 2015 to March 2010 were analyzed retrospectively. Their median age at diagnosis was 51 years old. 9/10 (90%) patients showed symptoms of anemia, with a median Hb level of 82.5 g/L, 5/10 (50%) patients combined with autoimmune disorders and with a median Hb level of 77 g/L. 7/10 (70%) patients had splenomegaly, 2/10 (20%) patients had complex karyotype, 2/10 (20%) patients had gene mutations, the median age of 4 patients with complex karyotype and gene mutation was 49 years old, all of them suffered from splenomegaly. The immunophenotype of 6/10 patients was CD3+ CD4- CD8+ and that of 2/10 patients (20%) was CD3+ CD4- CD8-, that of another 2/10 (20%) was CD3+ CD4+ CD8-, the clinical features between different types of immunization were not statistically different. T-LGLL patients often are old men, combined with anemia and splenomegaly, often associated with autoimmune diseases; the patients with complex karyotype and gene mutation are younger and they are more with hepatosplenomegaly; the guide role of different immunotypes for clinical strategy is no significant.