Maria dos Anjos Teixeira

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.

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.

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.

Aggressive mature natural killer cell neoplasms: report on a series of 12 European patients with emphasis on flow cytometry based immunophenotype and DNA content of neoplastic natural killer cells

Abstract We report 12 cases of aggressive natural killer (NK) cell neoplasms diagnosed in Portugal, with emphasis on flow cytometry. Ten patients had extranodal NK/T cell lymphoma, nasal type and two had aggressive NK cell leukemia, and seven were men and five were women, with a median age of 50 years. NK cells brightly expressed the CD56 adhesion molecule and CD94 lectin type killer receptor and had an activation-related HLA-DR+ CD45RA+ CD45RO+ immunophenotype, in most cases. In contrast, dim CD16 expression was found in a minor proportion of cases, whereas CD57 and the CD158a and CD158e1 killer immunoglobulin-like receptors were negative. One-third of cases showed a hyperploid DNA content and nearly all had a very high S-phase proliferative rate. The phenotypic features of the neoplastic NK cells would suggest that they represent the transformed counterpart of the CD56 + bright NK cells that circulate in normal blood.

Pure Red Cell Aplasia Associated to Clonal CD8+ T-Cell Large Granular Lymphocytosis: Dependence on Cyclosporin A Therapy

This case report details a single patient with pure red cell aplasia (PRCA) associated with clonal CD3+, TCRαβ+, TCR-Vβ8+, CD8+, CD57+ large granular lymphocytosis whose anaemia did not respond to conventional immunosuppressive therapy but did respond to cyclosporin A (CsA). The patient has become dependent on CsA for 7 years in order to control anaemia due to associated PRCA.

Guess what: Chronic 13q14.3+/CD5−/CD23+ lymphocytic leukemia in blood and t(11;14)(q13;q32)+/CD5+/CD23− mantle cell lymphoma in lymph nodes!

We report a case of a patient with two B‐cell lymphoproliferative disorders: CD5−/CD23+ B‐cell chronic lymphocytic leukemia and CD5+/CD23− mantle cell lymphoma. These disorders were diagnosed simultaneously based on flow cytometry, immunohistochemistry, fluorescence in situ hybridization, and polymerase chain reaction–based molecular studies. The B‐cell lymphocytic leukemia clone predominated in the blood and bone marrow, whereas the mantle cell clone predominated in lymph nodes. Cytometry Part B (Clin. Cytometry) 51B:41–44, 2003.

Aggressive natural-killer cell lymphoma presenting with skin lesions, breast nodule, suprarenal masses and life-threatening pericardial and pleural effusions.

We report the clinical and laboratory findings of a patient with an aggressive Epstein-Barr virus positive CD2+/CD56+ natural killer-cell lymphoma with a high mitotic activity and complex chromosomal abnormalities presenting with life-threatening pericardial and pleural effusions, disseminated skin lesions, breast nodule and large suprarenal masses. The clinical course was characterized by resistance to chemotherapy and relapsing pericardial and pleural effusions with respiratory and haemodynamic failure. Death occurred 4 months after the first manifestations of the disease as a consequence of cardiac tamponade.

Atopic Dermatitis-Like Non-Erythrodermic Leukemic Variant of CD3−/+dim CD4+ Cutaneous T-Cell Lymphoma Preceded by Cutaneous Papular Xanthomatosis

We report a patient with cutaneous papular xanthomatosis who 4 years later developed a CD3−/+dim/CD4+ T-cell lymphoma. Pruritic xerotic non-erythrodermic skin, eosinophilia and hyper-IgE were present and erroneously classified as atopic dermatitis. Flow cytometry and DNA ploidy analysis of both blood and skin lymphocytes, skin histology and blood T-cell receptor gene rearrangement studies confirmed diagnosis of T-cell lymphoma. Monoclonal CD3−/+dim/CD4+ T-cells were especially prone to the synthesis of IL-13, a cytokine that is involved in IgE-secretion, and comprised both a medium (diploid) and large (hyperploid) sized T-cell populations with a similar immunophenotype. The majority of the normal residual T-cells were large granular lymphocytes, expressed activation-related and natural-killer -associated markers and secreted high levels of interferon gamma, suggesting that they might correspond to active cytotoxic cells directed against the neoplastic T-lymphocytes.