Can peripheral T-cell lymphomas be morphologically subclassified? A morphometric approach to 21 cases

Morphometric analysis of nuclear sizes and shapes was carried out on semithin sections of lymph node for 21 patients suffering from non-Hodgkin's peripheral T-cell malignant lymphoma (ML) (excluding mycosis and Sézary syndrome). Twenty cases of B-cell ML and three cases of Sézary syndrome with massive lymph node infiltration were also studied as references. Wright and Isaacson's recent proposals were applied to classify the peripheral T-cell MLs into monomorphic medium-cell ML (eight cases), pleomorphic ML (nine cases), and monomorphic large-cell ML (four cases). These three classes were readily distinguishable by morphometric analysis of nuclear sizes. Nuclear areas and their coefficients of variation were higher in pleomorphic MLs than in monomorphic medium-cell MLs (p less than 0.01). Large-cell monomorphic MLs were set apart by the histograms of their nuclear sizes. The mitoses were evaluated on histological sections and found to be more numerous in pleomorphic ML than in monomorphic medium-cell ML (p less than 0.05). Nuclear irregularity in the 21 cases of peripheral T-cell ML was lower than in Sézary cells. Morphometry clearly demonstrates the morphological distinctiveness of the subclasses of peripheral T-cell ML. Their biological significance has yet to be determined.     

Subpopulations of human T lymphocytes. IV. Quantitation and distribution in patients with mycosis fungoides and Sézary syndrome.

T cell subpopulations (Tμ and Tγ cells) were examined in the peripheral blood from fourteen patients with mycosis fungoides and Sézary syndrome. One patient with Sézary syndrome having low lymphocyte count had higher proportions of Tγ cells when compared to controls while the other with high lymphocyte count (75% Sézary cells) lacked Tγ cells and had normal proportions of Tμ cells. T cells from a third patient with Sézary syndrome having high lymphocyte count (95% Sézary cells) lacked almost completely both Tμ and Tγ cells. Three of eleven patients with mycosis fungoides had a high proportion of Tγ cells and one had a high proportion of Tμ cells. Study of T cells in the peripheral blood, lymph nodes, and bone marrow from two patients with mycosis fungoides demonstrated that the quantitative abnormality of tμ and Tγ cells is shared by the peripheral blood and bone marrow and not by the lymph nodes. Heterogeneity of T cells subsets in mycosis fungoides appears to be in non-malignant T cells. However, in Sézary syndrome malignant Sézary T cells demonstrate heterogeneity with regard to receptors for IgM (Tμ) and IgG (Tγ).     

SC5 mAb represents a unique tool for the detection of extracellular vimentin as a specific marker of Sezary cells

Circulating malignant Sézary lymphocytes result from a clonal proliferation of memory/activated CD4(+)CD45RO(+) T lymphocytes primarily involving the skin. Recently, the CD158k/KIR3DL2 cell surface receptor has been identified to phenotypically characterize these cells. We previously described a mAb termed SC5 that identifies an unknown early activation cell membrane molecule. It is expressed selectively by T lymphocytes isolated from healthy individuals upon activation, and by circulating Sézary syndrome lymphocytes. In addition, we found that SC5 mAb was reactive with all resting T lymphocytes once permeabilized, indicating that SC5 mAb-reactive molecule might present distinct cellular localization according to the T cell activation status. In this study, we show for the first time that SC5 mAb recognizes the intermediate filament protein vimentin when exported to the extracellular side of the plasma membrane of viable Sézary malignant cells. We demonstrate that SC5 mAb is unique as it reacts with both viable malignant lymphocytes and apoptotic T cells. As vimentin is also detected rapidly at the cell membrane surface after normal T lymphocyte activation, it suggests that its extracellular detection on Sézary cells could be a consequence of their constitutive activation status. Finally, as a probable outcome of vimentin cell surface expression, autoantibodies against vimentin were found in the sera of Sézary syndrome patients.     

Malignant and nonmalignant T cell lines from human T cell lymphotropic virus type I-negative patients with Sézary syndrome.

The establishment of an in vitro model for cutaneous T cell lymphomas and Sézary syndrome has been difficult since T cells from individuals with these diseases do not proliferate in response to T cell mitogens. We found that conditioned media, collected from mitogen-activated PBMC from Sézary patients, contain an IL-2 receptor inducing factor. Despite their ostensible proliferative disorder, using a combination Sézary cell-conditioned media and rIL-2, we established IL-2 responsive, human T cell lymphotropic virus type I negative T cell lines from 23 patients, nine of which contain cells with the structural and/or genetic characteristics of neoplastic Sézary T cells.     

Interdigitating reticulum cells in lymph nodes of Sézary syndrome. Freeze-fracture and ultrathin-section morphology

Lymph nodes with extensive leukemic infiltration from three patients with the Sézary syndrome were examined in ultrathin sections and in freeze-fracture replicas. Sézary cells (SC) and interdigitating reticulum cells (IDC) were the predominant cell types in the lymph nodes. Both were closely connected with each other by apparently interdigitating cytoplasmic processes. The projections between these cells were, in the main, processes from the IDC. In freeze-fracture replicas these cellular processes did not appear as interdigitations but were more bubble-like, and for this reason these cells are imprecisely described by the term "interdigitating." The SC were seen to possess only short cytoplasmic processes. The frequent polar grouping of cell organelles in SC in the region of the contact zone with IDC and the high organelle content of IDC ('activated IDC') could be the morphologic expression of intense interaction between IDC and SC. IDC displayed three features in freeze-fracture which are not specific to the Sézary syndrome, but should be applicable to IDC in general: (1) they exhibited an approximately equal density of intramembrane particles in both the E-face and the P-face, (2) some of the intramembrane particles in the P-face were assembled in clusters and (3) the surface showed bubble-like formations of the cytoplasmic processes. On the basis of these properties it was possible to distinguish IDC from macrophages and lymphocytes in freeze-fracture replicas.     

A condition resembling pagetoid reticulosis in a laboratory mouse.

A raised, hairless cutaneous nodule was found incidentally at necropsy of a 24-month-old CD1 mouse. Histologically there was infiltration of the epidermis by a monomorphic population of moderate to large lymphocytoid cells. Many large cells had bizarre convolutions of the nuclear membrane and resembled the so-called Sézary or mycosis cells seen in epidermotropic T-cell proliferative disorders. The pattern of cutaneous involvement and the presence of the large cells with convoluted nuclei is characteristic of pagetoid reticulosis. No previous reports of such cutaneous lymphoid neoplasms in mice were found in an extensive literature search.     

Differences in electrophoretic mobility and cytochemistry of leukemic T-cells with identical immunologic phenotype (CD1 positive)

Leukaemic cells of acute T-lymphoblastic leukaemia and T-lymphoblastic lymphoma with secondary leukaemic course of disease which showed the same immunological phenotype (cortical thymocytes, thymocyte stage II), could be further differentiated according to their cytochemical pattern and electrophoretic mobility (EPM). Leukaemic cells of T-ALL usually reveal a high EPM, whereas EPM of leukaemic cells of T-lymphoblastic lymphoma was significantly lowered. In cytochemical respect acid phosphatase was positive in both cases of leukaemia. An activity of acid esterase, however, could only be demonstrated in leukaemic cells of T-lymphoblastic lymphoma. The findings are interpreted as manifestation of a different stage of maturity of both T-cell clones with the same immunological phenotype.     

Human T-cell lymphotropic virus type I and adult T-cell leukemia/lymphoma outside Japan and the Caribbean Basin

Ninety-six patients with the diagnosis of adult T-cell leukemia/lymphoma (ATLL) were identified in countries outside Japan and the Caribbean Basin. Seventy-four of these patients were initially diagnosed in the United States; 25 of 52 patients whose places of birth were known had been born in the United States. The detection of 14 patients born in the southeastern United States, all black, indicates a group deserving particular attention for studies of human T-cell lymphotropic virus type I (HTLV-I), a suspected etiologic agent in most cases of ATLL. Although geographic clustering of ATLL in areas endemic for HTLV-I, particularly southwest Japan and the Caribbean Basin, is a dramatic feature of this disease, a review of the literature indicates that HTLV-I-associated ATLL probably occurs sporadically in a much wider distribution, the disease being diagnosed in native-born African, Chinese, European, and Latin American patients. A registry for ATLL cases is suggested, to assist in the identification of risk factors for this disease and, at the same time, improve case definitions and early diagnosis.     

Reduced Level of the BCL11B Protein Is Associated with Adult T-Cell Leukemia/Lymphoma

Background

Adult T-cell leukemia/lymphoma (ATLL) develops in a small proportion of human T-cell leukemia virus type I (HTLV-I)-infected individuals. However, the mechanism by which HTLV-I causes ATLL has not been fully elucidated. To provide fundamental insights into the multistep process of leukemogenesis, we have mapped the chromosomal abnormalities in 50 ATLL cases to identify potential key regulators of ATLL.

Results

The analysis of breakpoints in one ATLL case with the translocations t(14;17)(q32;q22-23) resulted in the identification of a Kruppel zinc finger gene, BCL11B, which plays a crucial role in T-cell development. Among the 7 ATLL cases that we examined by immunofluorescence analysis, 4 displayed low and one displayed moderate BCL11B signal intensities. A dramatically reduced level of the BCL11B protein was also found in HTLV-I-positive T-cell lines. The ectopic expression of BCL11B resulted in significant growth suppression in ATLL-derived cell lines but not in Jurkat cells.

Conclusions

Our genetic and functional data provide the first evidence that a reduction in the level of the BCL11B protein is a key event in the multistep progression of ATLL leukemogenesis.     

Ras signaling contributes to survival of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Tax-positive T-cells

Ras signaling pathways play an important role in cellular proliferation and survival, and inappropriate activation of Ras frequently results in cell transformation and cancer. Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the etiological agent of the adult T-cell leukemia/lymphoma (ATLL), a severe malignancy that has a poor prognosis and exhibits resistance to conventional chemotherapy. Although the mechanisms involved in cell transformation by HTLV-1 have not been completely clarified, it is generally thought that Tax plays a pivotal role in the process. We have previously proposed that a functionally active Ras protein is needed for efficient anti-apoptotic activity of Tax. In this study we report data indicating that the apoptotic resistance of cells expressing Tax, constitutively or transiently, is linked to the intracellular levels of Ras-GTP. Indeed, we found that Tax-positive cells have a high content of active Ras, and that inhibition of Ras signaling, using the antagonist farnesyl thyosalicylic acid (FTS), increases their sensitivity to apoptosis. FTS treatment was also accompanied by a decrease in ERK, but not Akt, phosphorylation. Thus, all together our data suggest that the interaction between Tax and Ras could be important to ATLL pathogenesis, and indicate Ras as a possible target for therapeutic intervention in ATLL patients.     

Stem cell properties and the side population cells as a target for interferon-alpha in adult T-cell leukemia/lymphoma

The cancer stem cell theory suggests that chemoresistance and recurrence of tumors are often due to the similarity of stem cell properties between normal and cancer cells. Adult T-cell leukemia/lymphoma (ATLL) has poor prognosis, suggesting that ATLL cells possess common stem cell properties. We analyzed side population (SP), a characteristic stem cell phenotype, and CD markers in ATLL cell lines. We found that several lines contained SP with expressions of some hematopoietic stem cell markers. On the other hand, treatment with interferon (IFN)-α is sometimes effective in ATLL, particularly combined with other drugs. We examined its effect on ATLL cells and found that IFN-α significantly reduced the SP proportion. Moreover, CD25-positive cells and phosphorylation of STAT1/5 and ERK were upregulated during this process. These data suggest that their stem cell properties render ATLL cells therapy-resistant, and IFN-α exerts its clinical effect through a reduction of the SP cell population.     

Characterization of the immunophenotype and the metastatic properties of a murine T-lymphoma cell line. Unexpected expression of cytoplasmatic CD4

We report the first characterization of a mouse T-lymphoma cell line that surprisingly expresses cytoplasmatic (cy) yCD4. Phenotypically, LBC cells are CD5+, CD8+, CD16+, CD24+, CD25+, CD2-/dim, CD3-/dim, TCRbeta-/dim, TCRgammadelta, CD154 , CD40-, and CD45R. Coexpress cyTCRbeta, cyCD3, cyCD4, and yet lack surface CD4 expression. Transplantation of LBC cells into mice resulted in an aggressive T-lymphoblastic lymphoma that infiltrated lymph nodes, thymus, spleen, liver, ovary, and uterus but not peripheral blood or bone marrow. LBC cells display a modal chromosome number of 39 and a near-diploid karyotype. Based on the characterization data, we demonstrated that the LBC cell line was derived from an early T-cell lymphocyte precursor. We propose that the malignant cell transformation of LBC cells could coincide with the transition stage from late double-negative, DN3 (CD4- CD8 CD44-/low, CD25+) or DN4 (CD4-low, CD8-/low, CD44-, CD25-) to double-positive (DP: CD4+CD8+) stage of T-cell development. LBC cells provide a T-lymphoblastic lymphoma model derived from a malignant early T-lymphocyte that can be potentially useful as a model to study both cellular regulation and differentiation of T-cells. In addition, LBC tumor provides a short latency neoplasm to study cellular regulation and to perform preclinical trials of lymphoma-relatel clisorders.     

The regulation of NDRG2 expression during ATLL development after HTLV-1 infection

N-myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor that is frequently downregulated in adult T-cell leukemia/lymphoma (ATLL) and functions to negatively regulate several cellular signaling pathways as PP2A recruiter. To clarify the molecular mechanisms of suppression of NDRG2 expression, we initially determined the expression pattern of NDRG2 in various types of T-cells and ATLL cells. NDRG2 expression was significantly upregulated in HTLV-1/Tax-immortalized T-cells, which was mediated by NF-κB activation through Tax expression. On the other hand, NDRG2 expression was suppressed in HTLV-1-infected cell lines and various types of ATLL cells, which was dependent on the DNA methylation of the NDRG2 promoter. We found that the expression of enhancer of zeste homolog 2 (EZH2), a member of the polycomb family, is increased in ATLL, and that EZH2 directly binds to the NDRG2 promoter and induces DNA methylation of the NDRG2 promoter. Since the expression of EZH2 were anti-parallelly regulated with the NDRG2 expression, EZH2 might be one of the most important regulators of the downregulation of NDRG2, contributing to enhanced activation of signaling pathways during ATLL development.     

Celecoxib disrupts the canonical apoptotic network in HTLV-I cells through activation of Bax and inhibition of PKB/Akt

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive lymphoproliferative disease of very poor clinical prognosis associated with infection by the human T-cell leukemia virus type I (HTLV-I). Treatment of patients with ATLL using conventional chemotherapy has limited benefit because HTLV-I cells are refractory to most apoptosis-inducing agents. In this study, we report that Celecoxib induces cell death via the intrinsic mitochondrial pathway in HTLV-I transformed leukemia cells. Treatment with Celecoxib was associated with activation of Bax, decreased expression of Mcl-1, loss of the mitochondrial membrane potential and caspase-9-dependent apoptosis. These effects were independent from Bcl-2 and Bcl-xL. We also found that Celecoxib inhibited the Akt/GSK3 β survival pathway in HTLV-I cells. U. Sinha-Datta and J. M. Taylor have contributed equally to this work.     

WRN-targeted therapy using inhibitors NSC 19630 and NSC 617145 induce apoptosis in HTLV-1-transformed adult T-cell leukemia cells

Background

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATLL), a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the “Werner syndrome ATP-dependent helicase” encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells.

Methods

Our analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential.

Results

Targeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor) induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145.

Conclusions

WRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.

     

The significance of cytomegalovirus infection over the clinical course of adult T-cell leukemia/lymphoma

The significance of cytomegalovirus (CMV) infections developed over the clinical course of adult T-cell leukemia/lymphoma (ATLL) were evaluated in relation to the patient survival rate, ATL activity and immunocompetent cells. ATLL patients with CMV infections on admission exhibited a poor survival rate, while patients with CMV infections at any time after admission survived longer than those not infected with this virus. ATLL patients who exhibited a numbers of CMV infection on admission showed higher ATL activity and had lower numbers of CD8-positive and CD56-positive cells than those who developed CMV infections at any time after admission. Therefore, it appears likely that patients with CMV infections on admission were in an immunosuppressive state due to aggressive ATL activity.