CITÓMICA. Artículos del Grupo de Investigación CITÓMICA

Tγδ LGLL identifies a subset with more symptomatic disease: analysis of an international cohort of 137 patients

Sun, 01 Jan 2023 00:00:00 GMT

[EN]Tγδ large granular lymphocyte leukemia (LGLL) is a rare variant of T-cell LGLL (T-LGLL) that has been less investigated as compared with the more frequent Tαβ LGLL, particularly in terms of frequency of STAT3 and STAT5b mutations. In this study, we characterized the clinical and biological features of 137 patients affected by Tγδ LGLL; data were retrospectively collected from 1997 to 2020 at 8 referral centers. Neutropenia and anemia were the most relevant clinical features, being present in 54.2% and 49.6% of cases, respectively, including severe neutropenia and anemia in ∼20% of cases each. Among the various treatments, cyclosporine A was shown to provide the best response rates. DNA samples of 97 and 94 cases were available for STAT3 and STAT5b mutation analysis, with 38.1% and 4.2% of cases being mutated, respectively. Clinical and biological features of our series of Tγδ cases were also compared with a recently published Tαβ cohort including 129 cases. Though no differences in STAT3 and STAT5b mutational frequency were found, Tγδ cases more frequently presented with neutropenia (P = .0161), anemia (P < .0001), severe anemia (P = .0065), and thrombocytopenia (P = .0187). Moreover, Vδ2− cases displayed higher frequency of symptomatic disease. Overall, Tγδ cases displayed reduced survival with respect to Tαβ cases (P = .0017). Although there was no difference in STAT3 mutation frequency, our results showed that Tγδ LGLL represents a subset of T-LGLL characterized by more frequent symptoms and reduced survival as compared with Tαβ LGLL.

High frequency of low-count monoclonal B-cell lymphocytosis in hospitalized COVID-19 patients

Sun, 01 Jan 2023 00:00:00 GMT

[EN]Low-count monoclonal B-cell lymphocytosis (MBLlo) is associated with clinical and biological features typical of an underlying immunodeficiency, which may be a risk factor for developing more severe infections. Here we studied the frequency of MBLlo among COVID-19 patients and its potential impact on disease severity. The presence of MBLlo was investigated in blood of 249 COVID-19 patients vs. a pre-COVID-19 cohort of 728 individuas, using high-sensitivity flow cytometry; plasma levels of anti-SARS-CoV-2 antibodies were measured by ELISA. Overall, MBLlo was detected in 29% of COVID-19 patients (vs. 14% in the general pre-COVID-19 population; p<0.0001), particularly among hospitalized patients (40%; p<0.0001), with a tendency toward a predominance in men. MBLlo patients showed features of more severe COVID-19 than non-MBL cases, including more frequent (85% vs. 60%; p=0.0001) and longer (14 vs. 10 days; p=0.03) hospitalization, higher oxygen therapy (70% vs. 39%; p<0.0001) and need for intensive care (15% vs. 6%; p=0.02). Except for lower B-cell counts at recovery in MBLlo cases (p=0.04), similarly altered immune-cell profiles were observed in patients with and without MBLlo. In contrast, MBLlo patients showed a significantly higher percentage of detectable anti-SARS-CoV-2 IgM/IgG/IgA antibodies in plasma, associated with higher IgA SARS-CoV-2 specific antibody levels during acute infection. In conclusion, MBLlo is significantly more prevalent in COVID-19 patients vs. the general population, and it is associated with more severe COVID-19, further supporting the existence of a pre-established immunodeficiency already at early MBL stages.

Maturation-Related and Functional-Associated Phenotypic Profile of Tumor T Cells in Mature/Peripheral T-Cell Neoplasms: Association With the Diagnostic Subtype of the Disease

Wed, 01 Jan 2025 00:00:00 GMT

[EN]T-cell chronic lymphoproliferative disorders (T-CLPD) are a heterogeneous group of mature T-cell malignancies, the classification of which remains challenging. In this study, we classified tumor cells from 86 patients diagnosed with either T-CLPD (n = 81) or T-cell acute lymphoblastic leukemia (n = 5) into precise functional and maturation-associated compartments, based on their phenotypic similarities with their normal maturation-related and functional associated T-cell counterparts. A database was generated using blood samples from 6 sex- and age-matched healthy donors as a template for normal T-cell subset flow cytometric immunophenotypes, to which tumor cells of individual patients were compared. Except for nodal T follicular-helper cell lymphoma and adult T-cell leukemia/lymphoma, which showed phenotypes overlapping with that of T follicular-helper and T regulatory cells, respectively, all other T-CLPD displayed immunophenotypic profiles consistent with conventional T helper (Th) cells, with different maturation-associated profiles per diagnostic category. These included predominant naive/naive-central memory phenotypes in T-cell prolymphocytic leukemia to terminal effector cytotoxic cellular profiles in T-cell large granular lymphocytic leukemia; other T-CLPD diagnostic categories (mostly Sézary syndrome/mycosis fungoides) resembled the diverse memory T-cell subsets. Interestingly, immunophenotypically less-mature tumor cells (T-cell prolymphocytic leukemia) displayed more heterogeneous Th profiles, whereas those with memory T-cell profiles showed more consistent Th-associated patterns (eg, Th2 or Th17 in Sézary syndrome/mycosis fungoides), and the most mature neoplasms (eg, T-cell large granular lymphocytic leukemia) systematically displayed a Th1-like pattern, reflecting progressively lower plasticity for the more advanced tumor-associated maturation stages. These findings confirm the presence of distinct phenotypic patterns resembling specific maturation-associated and Th-related profiles of normal T cells among distinct diagnostic categories of T-CLPD, which might contribute to a more precise classification of T-CLPD.

Sézary syndrome and mycosis fungoides: An overview, including the role of immunophenotyping

Fri, 01 Jan 2021 00:00:00 GMT

[EN]This 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.

Age Distribution of Multiple Functionally Relevant Subsets of CD4+ T Cells in Human Blood Using a Standardized and Validated 14-Color EuroFlow Immune Monitoring Tube

Wed, 01 Jan 2020 00:00:00 GMT

[EN]CD4+ T cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sensitive and reproducible flow cytometry monitoring of CD4+ T-cell populations in human blood to establish normal age-related reference values and evaluate the presence of potentially altered profiles in three distinct disease models-monoclonal B-cell lymphocytosis (MBL), systemic mastocytosis (SM), and common variable immunodeficiency (CVID). Overall, 145 blood samples from healthy donors were used to design and validate a 14-color antibody combination based on extensive reagent testing in multiple cycles of design-testing-evaluation-redesign, combined with in vitro functional studies, gene expression profiling, and multicentric evaluation of manual vs. automated gating. Fifteen cord blood and 98 blood samples from healthy donors (aged 0-89 years) were used to establish reference values, and another 25 blood samples were evaluated for detecting potentially altered CD4 T-cell subset profiles in MBL (n = 8), SM (n = 7), and CVID (n = 10). The 14-color tube can identify ≥89 different CD4+ T-cell populations in blood, as validated with high multicenter reproducibility, particularly when software-guided automated (vs. manual expert-based) gating was used. Furthermore, age-related reference values were established, which reflect different kinetics for distinct subsets: progressive increase of naïve T cells, T-helper (Th)1, Th17, follicular helper T (TFH) cells, and regulatory T cells (Tregs) from birth until 2 years, followed by a decrease of naïve T cells, Th2, and Tregs in older children and a subsequent increase in multiple Th-cell subsets toward late adulthood. Altered and unique CD4+ T-cell subset profiles were detected in two of the three disease models evaluated (SM and CVID). In summary, the EuroFlow immune monitoring TCD4 tube allows fast, automated, and reproducible identification of ≥89 subsets of CD4+ blood T cells, with different kinetics throughout life. These results set the basis for in-depth T-cell monitoring in different disease and therapeutic conditions.

STAT3 and STAT5B Mutations in T/NK-Cell Chronic Lymphoproliferative Disorders of Large Granular Lymphocytes (LGL): Association with Disease Features

Wed, 25 Nov 2020 00:00:00 GMT

[EN]STAT3 and STAT5B (STAT3/STAT5B) mutations are the most common mutations in T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorders of NK cells (CLPD-NK), but their clinical impact remains unknown. We investigated the frequency and type of STAT3/STAT5B mutations in FACS-sorted populations of expanded T/NK-LGL from 100 (82 clonal; 6 oligoclonal; 12 polyclonal) patients, and its relationship with disease features. Seventeen non-LGL T-CLPD patients and 628 age-matched healthy donors were analyzed as controls. STAT3 (n = 30) and STAT5B (n = 1) mutations were detected in 28/82 clonal T/NK-LGLL patients (34%), while absent (0/18, 0%) among oligoclonal/polyclonal LGL-lymphocytosis. Mutations were found across all diagnostic subgroups: TCD8+-LGLL, 36%; CLPD-NK, 38%; TCD4+-LGLL, 7%; Tαβ+DP-LGLL, 100%; Tαβ+DN-LGLL, 50%; Tγδ+-LGLL, 44%. STAT3-mutated T-LGLL/CLPD-NK showed overall reduced (p < 0.05) blood counts of most normal leukocyte subsets, with a higher rate (vs. nonmutated LGLL) of neutropenia (p = 0.04), severe neutropenia (p = 0.02), and cases requiring treatment (p = 0.0001), together with a shorter time-to-therapy (p = 0.0001), particularly in non-Y640F STAT3-mutated patients. These findings confirm and extend on previous observations about the high prevalence of STAT3 mutations across different subtypes of LGLL, and its association with a more marked decrease of all major blood-cell subsets and a shortened time-to-therapy.

Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell Clonality: Standardization of Sample Preparation and Diagnostic Implementation

Fri, 01 Jan 2021 00:00:00 GMT

[EN]A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1- monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1- ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1+/TRBC1- ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10-4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ+ leukemia/lymphoma patients.

Non-apoptotic FAS signaling controls mTOR activation and extrafollicular maturation in human B cells.

Fri, 12 Jan 2024 00:00:00 GMT

[EN]Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

Low-count monoclonal B-cell lymphocytosis persists after seven years of follow up and is associated with a poorer outcome

Sun, 01 Jul 2018 00:00:00 GMT

[EN]Low-count monoclonal B-cell lymphocytosis is defined by the presence of very low numbers of circulating clonal B cells, usually phenotypically similar to chronic lymphocytic leukemia cells, whose biological and clinical significance remains elusive. Herein, we re-evaluated 65/91 low-count monoclonal B-cell lymphocytosis cases (54 chronic lymphocytic leukemia-like and 11 non-chronic lymphocytic leukemialike) followed-up for a median of seven years, using high-sensitivity flow cytometry and interphase fluorescence in situ hybridization. Overall, the clone size significantly increased in 69% of low-count monoclonal B-cell lymphocytosis cases, but only one subject progressed to high-count monoclonal B-cell lymphocytosis. In parallel, the frequency of cytogenetic alterations increased over time (32% vs. 61% of cases, respectively). The absolute number of the major T-cell and natural killer cell populations also increased, but only among chronic lymphocytic leukemia-like cases with increased clone size vs. age- and sex-matched controls. Although progression to chronic lymphocytic leukemia was not observed, the overall survival of low-count monoclonal B-cell lymphocytosis individuals was significantly reduced vs. non-monoclonal Bcell lymphocytosis controls (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer were the main causes of death in low-count monoclonal B-cell lymphocytosis. In summary, despite the fact that mid-term progression from low-count monoclonal B-cell lymphocytosis to high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia appears to be unlikely, these clones persist at increased numbers, usually carrying more genetic alterations, and might thus be a marker of an impaired immune system indirectly associated with a poorer outcome, particularly among females.

Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma

Sun, 01 Oct 2017 00:00:00 GMT

[EN]Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics.

Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality

Fri, 06 Nov 2015 00:00:00 GMT

[EN]Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56(low) NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56(low) NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94(hi)/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality.