Margarida Lima

Immunophenotypic Analysis of the TCR-Vβ Repertoire in 98 Persistent Expansions of CD3+/TCR-αβ+ Large Granular Lymphocytes : Utility in Assessing Clonality and Insights into the Pathogenesis of the Disease

At present, a major challenge in the initial diagnosis of leukemia of large granular lymphocytes (LGLs) is to establish the clonal nature of the expanded population. In the present study we have analyzed by flow cytometry immunophenotyping the TCR-Vβ repertoire of 98 consecutive cases of persistent expansions of CD4 + or CD8 +bright CD3 + /TCR-αβ + LGLs and compared the results with those obtained in molecular studies of TCR-β gene rearrangements. Fifty-eight cases were considered to be monoclonal in molecular studies whereas in the remaining 40 cases there was no evidence for monoclonality (11 cases were considered oligoclonal and 29 polyclonal). The TCR-Vβ repertoire was biased to the preferential use of one or more TCR-Vβ families in 96% of cases, a total of 124 TCR-Vβ expansions being diagnosed: one TCR-Vβ expansion in 71 cases and two or more TCR-Vβ expansions in 23 cases. The highest TCR-Vβ expansion observed in each case was higher among monoclonal (74 ± 19%) as compared to nonmonoclonal cases (24 ± 14%) ( P = 0.001), as did the fraction of LGLs that exhibited a TCR-Vβ-restricted pattern (86 ± 16% and 42 ± 23%, respectively; P = 0.0001); by contrast, the proportion of cases displaying more than one TCR-Vβ expansion was higher in the latter group: 7% versus 48%, respectively ( P = 0.001). Results obtained in oligoclonal cases were intermediate between those obtained in polyclonal and monoclonal cases and similar results were observed for CD4 + as for CD8 +bright T-cell expansions. TCR-Vβ familiesexpressed in CD8 +bright T-cell-LGL proliferations showed a pattern of distribution that mimics the frequency at which the individual TCR-Vβ families are represented in normal peripheral blood T cells. Assuming that a given proliferation of LGLs is monoclonal whenever there is an expansion of a given TCR-Vβ family of at least 40% of the total CD4 + or CD8 +bright T-cell compartment, we were able to predict clonality with a sensitivity of 93% and a specificity of 80%. By increasing the cut-off value to 60%, sensitivity and specificity were of 81% and 100%. In summary, our results suggest that flow cytometry immunophenotypic analysis of the TCR-Vβ repertoire is a powerful screening tool for the assessment of T-cell clonality in persistent expansions of TCR-αβ + LGLs.

TCRαβ+/CD4+ large granular lymphocytosis: A new clonal T-cell lymphoproliferative disorder

Large granular lymphocyte (LGL) leukemia is a well-recognized disease of mature T-CD8+ or less frequently natural killer cells; in contrast, monoclonal expansions of CD4+ T-LGL have only been sporadically reported in the literature. In the present article we have explored throughout a period of 56 months the incidence of monoclonal expansions of CD4+ T-LGL in a population of 2.2 million inhabitants and analyzed the immunophenotype and the pattern of cytokine production of clonal CD4+ T cells of a series of 34 consecutive cases. Like CD8+ T-LGL leukemias, CD4+ T-LGL leukemia patients have an indolent disease; however, in contrast to CD8+ T-LGL leukemias, they do not show cytopenias and autoimmune phenomena and they frequently have associated neoplasias, which is usually determining the clinical course of the disease. Monoclonal CD4+ T-LGLshowed expression of TCRαβ, variable levels of CD8 (CD8−/+dim) and a homogeneous typical cytotoxic (granzyme B+, CD56+, CD57+, CD11b+/−) and activated/memory T cell (CD2+bright, CD7−/+dim, CD11a+bright, CD28−, CD62L− HLA-DR+) immunophenotype. In addition, they exhibited a Th1 pattern of cytokine production [interferon-γ++, tumor necrosis factor-α++, interleukin (IL-2)−/+, IL-4−, IL-10−, IL-13−]. Phenotypic analysis of the TCR-Vβ repertoire revealed large monoclonal TCR-Vβ expansions; only a restricted number of TCR-Vβ families were represented in the 34 cases analyzed. These findings suggest that monoclonal TCRαβ+/CD4+/NKa+/CD8−/+dim T-LGL represent a subgroup of monoclonal LGL lymphoproliferative disorders different from both CD8+ T-LGL and natural killer cell-type LGL leukemias. Longer follow-up periods are necessary to determine the exact significance of this clonal disorder.

Inflammation, T-Cell Phenotype, and Inflammatory Cytokines in Chronic Kidney Disease Patients Under Hemodialysis and its Relationship to Resistance to Recombinant Human Erythropoietin Therapy

BackgroundResistance to recombinant human erythropoietin (rhEPO) occurs in some chronic kidney disease (CKD) patients, which may be due to enhanced systemic inflammatory response and to the erythropoiesis-suppressing effect of pro-inflammatory cytokines, some of which are produced by T cells.Aim of studyThe aim of this study was to investigate the relationship between resistance to rhEPO therapy in hemodialysis CKD patients and inflammatory markers [C-reactive protein (CRP), soluble interleukin (IL)-2 receptor (sIL2R), and serum albumin levels], blood cell counts, T-cell phenotype, cytokine production by T cells, and serum cytokine levels.Materials and MethodsWe studied 50 hemodialysis CKD patients, 25 responders and 25 nonresponders to rhEPO, and compared them to each other and with 25 healthy controls. When compared to controls, CKD patients showed increased serum levels of CRP, IL-6, and sIL2R and a T-cell lymphopenia, due to decreased numbers of both CD4+ and CD8+ T cells. T cells from CKD patients had an immunophenotype compatible with chronic T-cell stimulation as shown by the increased percentage of CD28−, CD57+, HLA-DR+, CD28−HLA-DR+, and CD57+ HLA-DR+ T cells and produce higher levels of IL-2, INF-γ, and TNF-α after short-term in vitro stimulation, although Th1 cytokines were not detectable in serum. Statistically significant differences were found between responders and nonresponders to rhEPO therapy for total lymphocyte and CD4+ T-lymphocyte counts, albumin (lower in nonresponders) and CRP (higher in nonresponders) levels.ConclusionCKD patients under hemodialysis present with raised inflammatory markers and decrease of total lymphocyte and CD4+ T-lymphocyte counts when compared with controls. Some of those markers are even further enhanced in nonresponders to rhEPO therapy patients, but resistance to this therapy cannot be justified by a Th1 polarized T-cell response.

Regular ArticlesTCRαβ+/CD4+ Large Granular Lymphocytosis: A New Clonal T-Cell Lymphoproliferative Disorder

Large granular lymphocyte (LGL) leukemia is a well-recognized disease of mature T-CD8+ or less frequently natural killer cells; in contrast, monoclonal expansions of CD4+ T-LGL have only been sporadically reported in the literature. In the present article we have explored throughout a period of 56 months the incidence of monoclonal expansions of CD4+ T-LGL in a population of 2.2 million inhabitants and analyzed the immunophenotype and the pattern of cytokine production of clonal CD4+ T cells of a series of 34 consecutive cases. Like CD8+ T-LGL leukemias, CD4+ T-LGL leukemia patients have an indolent disease; however, in contrast to CD8+ T-LGL leukemias, they do not show cytopenias and autoimmune phenomena and they frequently have associated neoplasias, which is usually determining the clinical course of the disease. Monoclonal CD4+ T-LGLshowed expression of TCRαβ, variable levels of CD8 (CD8−/+dim) and a homogeneous typical cytotoxic (granzyme B+, CD56+, CD57+, CD11b+/−) and activated/memory T cell (CD2+bright, CD7−/+dim, CD11a+bright, CD28−, CD62L− HLA-DR+) immunophenotype. In addition, they exhibited a Th1 pattern of cytokine production [interferon-γ++, tumor necrosis factor-α++, interleukin (IL-2)−/+, IL-4−, IL-10−, IL-13−]. Phenotypic analysis of the TCR-Vβ repertoire revealed large monoclonal TCR-Vβ expansions; only a restricted number of TCR-Vβ families were represented in the 34 cases analyzed. These findings suggest that monoclonal TCRαβ+/CD4+/NKa+/CD8−/+dim T-LGL represent a subgroup of monoclonal LGL lymphoproliferative disorders different from both CD8+ T-LGL and natural killer cell-type LGL leukemias. Longer follow-up periods are necessary to determine the exact significance of this clonal disorder.

Clinicobiological, Immunophenotypic, and Molecular Characteristics of Monoclonal CD56−/+dim Chronic Natural Killer Cell Large Granular Lymphocytosis

Indolent natural killer (NK) cell lymphoproliferative disorders include a heterogeneous group of patients in whom persistent expansions of mature, typically CD56(+), NK cells in the absence of any clonal marker are present in the peripheral blood. In the present study we report on the clinical, hematological, immunophenotypic, serological, and molecular features of a series of 26 patients with chronic large granular NK cell lymphocytosis, whose NK cells were either CD56(-) or expressed very low levels of CD56 (CD56(-/+dim) NK cells), in the context of an aberrant activation-related mature phenotype and proved to be monoclonal using the human androgen receptor gene polymerase chain reaction-based assay. As normal CD56(+) NK cells, CD56(-/+dim) NK cells were granzyme B(+), CD3(-), TCRalphabeta/gammadelta(-), CD5(-), CD28(-), CD11a(+bright), CD45RA(+bright), CD122(+), and CD25(-) and they showed variable and heterogeneous expression of both CD8 and CD57. Nevertheless, they displayed several unusual immunophenotypic features. Accordingly, besides being CD56(-/+dim), they were CD11b(-/+dim) (heterogeneous), CD7(-/+dim) (heterogeneous), CD2(+) (homogeneous), CD11c(+bright) (homogeneous), and CD38(-/+dim) (heterogeneous). Moreover, CD56(-/+dim) NK cells heterogeneously expressed HLA-DR. In that concerning the expression of killer receptors, CD56(-/+dim) NK cells showed bright and homogeneous CD94 expression, and dim and heterogeneous reactivity for CD161, whereas CD158a and NKB1 expression was variable. From the functional point of view, CD56(-/+dim) showed a typical Th1 pattern of cytokine production (interferon-gamma(+), tumor necrosis factor-alpha(+)). From the clinical point of view, these patients usually had an indolent clinical course, progression into a massive lymphocytosis with lung infiltration leading to death being observed in only one case. Despite this, they frequently had associated cytopenias as well as neoplastic diseases and/or viral infections. In summary, we describe a unique and homogeneous group of monoclonal chronic large granular NK cell lymphocytosis with an aberrant activation-related CD56(-/+dim)/CD11b(-/+dim) phenotype and an indolent clinical course, whose main clinical features are related to concomitant diseases.

TCRγδ+ large granular lymphocyte leukemias reflect the spectrum of normal antigen-selected TCRγδ+ T-cells

T-cell large granular lymphocytes (LGL) proliferations range from reactive expansions of activated T cells to T-cell leukemias and show variable clinical presentation and disease course. The vast majority of T-LGL proliferations express TCRαβ. Much less is known about the characteristics and pathogenesis of TCRγδ+ cases. We evaluated 44 patients with clonal TCRγδ+ T-LGL proliferations with respect to clinical data, immunophenotype and TCR gene rearrangement pattern. TCRγδ+ T-LGL leukemia patients had similar clinical presentations as TCRαβ+ T-LGL leukemia patients. Their course was indolent and 61% of patients were symptomatic. The most common clinical manifestations were chronic cytopenias – neutropenia (48%), anemia (23%), thrombocytopenia (9%), pancytopenia (2%) – and to a lesser extent splenomegaly (18%). Also multiple associated autoimmune (34%) and hematological (14%) disorders were found. Leukemic LGLs were predominantly positive for CD2, CD5, CD7, CD8, and CD57, whereas variable expression was seen for CD16, CD56, CD11b, and CD11c. The Vγ9/Vδ2 immunophenotype was found in 48% of cases and 43% of cases was positive for Vδ1, reflecting the TCR-spectrum of normal TCRγδ+ T-cells in adult PB. Identification of the well-defined post-thymic Vδ2-Jδ1 selection determinant in all evaluable Vγ9+/Vδ2+ patients, is suggestive of common (super)antigen involvement in the pathogenesis of these TCRγδ+ T-LGL leukemia patients.

Aggressive mature natural killer cell neoplasms: from epidemiology to diagnosis

Mature natural killer (NK) cell neoplasms are classified by the World Health Organization into NK/T cell lymphoma, nasal type (NKTCL), aggressive NK-cellleukemia (ANKCL) and chronic lymphoproliferative disorders of NK-cells, thelatter being considered provisionally. NKTCL and ANKCL are rare diseases, withhigher prevalence in Asia, Central and South America. Most NKTCL presentextranodal, as a destructive tumor affecting the nose and upper aerodigestivetract (nasal NKTCL) or any organ or tissue (extranasal NKTCL) whereas ANKCLmanifests as a systemic disease with multiorgan involvement and naturallyevolutes to death in a few weeks. The histopathological hallmark of theseaggressive NK-cell tumors is a polymorphic neoplastic infiltrate withangiocentricity, angiodestruction and tissue necrosis. The tumor cells havecytoplasmatic azurophilic granules and usually show a CD45+bright,CD2+, sCD3-, cytCD3epsilon+,CD56+bright, CD16−/+, cytotoxic granulesmolecules+ phenotype. T-cell receptor genes are in germ-lineconfiguration. Epstein-Barr virus (EBV) -encoded membrane proteins and earlyregion EBV RNA are usually detected on lymphoma cells, with a pattern suggestiveof a latent viral infection type II. Complex chromosomal abnormalities arefrequent and loss of chromosomes 6q, 11q, 13q, and 17p are recurrentaberrations. The rarity of the NK-cell tumors limits our ability to standardizethe procedures for the diagnosis and clinical management and efforts should bemade to encourage multi-institutional registries.ResumoAs neoplasias de células natural killer (NK) maduras foramclassificadas pela Organização Mundial de Saúde em trêsentidades: o linfoma de células NK/T tipo nasal (NKTCL), a leucemiaagressiva de células NK (ANKCL) e as doenças linfoproliferativascrónicas de células NK, estas últimas consideradas umaentidade provisória. Os NKTCL e a ANKCL são doenças raras,mais prevalentes na Ásia, na América Central e na América doSul. A maioria dos NKTCL tem uma apresentação extra-ganglionar, naforma de tumor destrutivo que atinge o nariz e o trato aerodigestivo alto(forma nasal) ou qualquer órgão ou tecido (forma extranasal). AANKCL manifesta-se como uma doença sistémica que evolui para amorte em poucas semanas. Do ponto de vista histopatológico, estasneoplasias caraterizam-se por um infiltrado polimórfico, comangiocentricidade, destruição vascular e necrose tecidular. Ascélulas tumorais têm grânulos azurófilos no citoplasma eo seu imunofenótipo (CD45+forte, CD2+,sCD3-, cytCD3epsilon+, CD56+forte,CD16−/+, proteínas dos grânuloscitotóxicos+) é caraterístico. Os genes quecodificam para o recetor das células T estão emconfiguração nativa. As células tumorais expressam geralmenteproteínas da membrana e ARN do vírus Epstein Barr, com umpadrão sugestivo de uma infecção vírica latente tipo II.As alterações cromossómicas são complexas, e algumas,como deleções nos braços longos dos cromossomas 6, 11 e 13 edo braço curto do cromossoma 17, ocorrem de forma recorrente. Araridade dos tumores de células NK limita a nossa capacidade parauniformizar os procedimentos de diagnóstico e a abordagem clínica,sendo necessário desenvolver esforços para promover os registosmulticêntricos.

Chronic eosinophilic leukaemia presenting with erythroderma, mild eosinophilia and hyper‐IgE: clinical, immunological and cytogenetic features and therapeutic approach. A case report.

A 23-year-old, white male metallurgist presented with pruritic erythematous maculo-papules over the trunk and upper limbs and 6 months later developed erythroderma, eosinophilia and multi-organ dysfunction. A diagnosis of chronic eosinophilic leukaemia was made on the basis of myeloproliferative involvement of both peripheral blood and bone marrow, associated with eosinophilic differentiation and a t(5;12)(q33;p13) translocation. The initial therapeutic approach was interferon alfa-2b plus cytosine arabinoside, for 13 months, followed by hydroxyurea plus vincristine. There was improvement of skin lesions, disappearance of eosinophilia and decrease of serum immunoglobulin E, towards normal values.

Cytogenetic findings in a patient presenting simultaneously with chronic lymphocytic leukemia and acute myeloid leukemia

A case of simultaneous presentation of B-chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) is described. CLL was documented by bone marrow and peripheral blood lymphocytosis with a typical B-CLL immunophenotype. The diagnosis of AML was supported by the presence of bone marrow and circulating blast cells positive for myeloperoxidase and myeloid-associated markers. Although the immunophenotyping and morphocytochemical studies indicated two different cell populations (mature B-CLL lymphocytes and myeloblasts), chromosome aberrations commonly associated with CLL and AML were found simultaneously in the same metaphases obtained from unstimulated 24-hour cultures of peripheral blood cells.

The gel test: some problems and solutions

SUMMARY. The gel centrifugation test (GT) is a method of transfusion serology, based on the fact that, after centrifugation, unagglutinated red blood cells (RBC) pass easily through a gel, while agglutinated RBC do not. The introduction of the GT to our blood bank transfusion routine [strictly following the manufacturers instructions (DiaMed ID Micro Typing System)] resulted in problems with the interpretation of the results. These were overcome after the introduction of modifications, which included; (1) the systematic use of 1% RBC suspensions; (2) the use of 50 μ1 of 1% RBC suspensions and 25 μ1 of serum in all tests; (3) the control of all negative indirect antiglobulin tests (IAT) and direct antiglobulin tests (DAT) by the addition of 50 μ1 of a 1% IgG coated RBC suspension followed by centrifugation; and (4) the systematic use of saline‐suspended RBC for ABO typing in patients with positive DAT.