Identification of TCL1A as an immunohistochemical marker of adverse outcome in diffuse large B-cell lymphomas

We used a combination of DNA-microarray and tissue-microarray (TMA) analyses to identify markers that could be routinely used to predict the outcome of diffuse large-B-cell lymphoma (DLCL) patients. Gene expression profiling was performed using DNA-microarrays on 52 tumour biopsy samples [31 DLCL and 21 follicular lymphomas (FL)] from 48 patients (28 DLCL and 20 FL). T-cell leukemia/lymphoma-1A (TCL1A) mRNA overexpression was correlated with relapse in DLCL patients. TMA analysis was applied on a distinct series of 36 formalin-fixed, paraffin-embedded DLCL samples and showed that TCL1A immunoexpression was correlated with either higher relapse (p=0.02) or lower 5-year overall survival (p=0.009) rates. Moreover, the prognostic value of TCL1A was independent from IPI in our series. Our data suggest that TCL1A immunodetection is an independent marker of adverse outcome that could be used in routine settings for the management of DLCL patients.     

Detection of Epstein-Barr virus and subsets of lymphoid cells in adenoid tissue of children under 2 years of age

Epstein-Barr virus (EBV) has been closely associated with undifferentiated nasopharyngeal carcinoma (NPC) and T/NK nasal non Hodgkin lymphoma. Nevertheless, the presence of EBV in non neoplastic lymphoid tissue of the nasopharynx has been rarely investigated. In a previous study by our group, using in situ hybridization to detect EBV in adenoids of children (2-13 years old) resected because of nasal obstruction due to hypertrophy, we found EBV genome in 72% of the cases. It was now intended to study the frequency of EBV expression in adenoids from children that underwent surgical removal, belonging to a lower age group (1-2 years old). It was also intended to establish which lymphoid subsets are involved in this infection. Adenoidal paraffin sections from 21 patients aged 1-2 years old (mean 1.6 years), 15 males and six females were submitted to double labeling: in situ hybridization with EBER 1/2 probes to detect EBV and immunohistochemistry to determine the lymphocyte typing of EBV-positive cells (CD20 for B-lymphocytes, CD3 for T-lymphocytes and CD56 and CD57 for NK-cells). Among 21 patients, seven showed positive lymphoid cells for EBV (33%). In almost all cases, EBV-positive cells were also CD20-positive. Some EBV-positive cells showed no labeling with any of the lymphoid markers, but in no instance they were positive for CD3, CD56 or CD57. This study confirms the preferential infection of B-lymphocytes by EBV, which in some instances can down regulate the expression of CD20.     

Production of anti-B monoclonal antibodies (DBB.42, DBA.44, DNA.7, and DND.53) reactive on paraffin-embedded tissues with a new B-lymphoma cell line grafted into athymic nude mice

A new B-lymphoma cell line (DEAU-cell line) was established from a diffuse large-cell lymphoma (centroblastic type) and was successfully grafted in athymic nude mice. Monoclonal antibodies (MoAbs) were generated using splenocytes of DEAU-tumor bearing mice. Before the fusion experiments, cellular immunity of the mice bearing growing DEAU tumors was restored by injection of spleen cells from conventional Balb/C mice. Spleen cells from conventional Balb/C mice immunized with DEAU-cell line were also used for the generation of MoAbs. Four MoAbs (DBB.42 and DBA.44 from normal Balb/C mice, and DNA.7 and DND.53 from athymic nude mice) were investigated because they identified B-cell- associated antigens not destroyed by fixatives. DBB.42 recognized a pan- B cell-associated antigen (molecular weight (mol wt) = 45 Kd). DBA.44 detected a B-cell antigen (mol wt not determined) expressed on a subpopulation of B lymphocytes in the mantle zone of lymphoid follicles. DNA.7 also defined a B-cell antigen (43 Kd) mainly expressed on germinal center cells. Similarly, DND.53 recognized a B-cell antigen (two bands of mol wt 20 Kd and 35 Kd, respectively) mainly expressed on germinal center cells and mantle zone lymphocytes and interdigitating reticulum cells in the paracortical area. Major differences were found in the reactivities of these MoAbs on malignant lymphomas. DBB.42 was positive with almost all B-cell lymphomas and some T-cell lymphomas. Within the group of low-grade B-cell lymphomas, DBA.44 reacted principally with hairy-cell leukemia. DNA.7 reacted mainly with high- grade B-cell lymphomas with a weak positivity in low-grade B-cell lymphomas. DND.53 reacted with all but one B-cell lymphoma, cells of histiocytosis X, and Reed-Sternberg cells. These findings indicate that new MoAbs can be generated by using spleen cells from athymic mice bearing human tumors as well as by new lymphoid cell lines. The MoAbs so generated, as in the present study, are deemed potentially useful for the recognition of B-cell lymphomas in routine diagnostic histopathology. In addition, DND.53 could be of value for the diagnosis of histiocytosis X and the detection of Reed-Sternberg cells in Hodgkin's disease.     

High expression of the bcl-x gene in Reed-Sternberg cells of Hodgkin's disease

The expression of bcl-x protein, a bcl-2-related protein present in cortical thymocytes, activated lymphocytes, and plasma cells of reactive lymph nodes, was investigated in 44 cases of Hodgkin's disease (HD) in parallel with bcl-2 and Epstein-Barr virus (EBV) status. Eighty- six percent of the cases were positive for bcl-x, among them 27% with a strong signal in more than 75% of the Reed-Sternberg cells. Positivity for bcl-x was found in, respectively, 100% and 92% of the nodular sclerosis and mixed cellularity subtypes, although 4 cases of lymphocyte predominance subtype were negative. This finding was in contrast with the weaker positivity for bcl-2 staining in 44% of the cases. EBV small RNAs were detected in 43% of the cases by using in situ hybridization. Of interest, 100% of the EBV-positive samples were positive for bcl-x, whereas only 38% of these cases were bcl-2 positive. Our findings show that the bcl-x gene expression is high in HD, suggesting that bcl-x may have a role in the pathogenesis of at least some cases of HD via apoptosis regulation.     

Quantitative study of KI-67 antibody staining in 46 T-cell malignant lymphomas using image analysis.

Image analysis with a SAMBA 2005 (ALCATEL-TITN, Co) was used to quantify the Ki-67 stained area percentage in 46 T-cell malignant lymphomas (T-ML), classified according to the updated Kiel classification. This parameter demonstrated correlation with the number of Ki-67-positive cellular profiles (r = 0.88, P less than 0.001) and was more reproducible than cell counting. A significant difference was found between low and high grade T-ML (mean values +/- SEM respectively of 10.20 +/- 1.82 per cent and 25.63 +/- 3.15 per cent). The most interesting findings were that: (1) AILD-type T-ML showed an intermediate proliferation rate (15.55 +/- 2.72 per cent) between pleomorphic T-ML with medium and with large cells (respectively 12.53 +/- 3.64 per cent and 22.43 +/- 3.46 per cent), both of which belong to the high grade malignancy group. This finding is in accordance with the poor prognosis of this subtype despite its classification in the low grade malignancy group. (2) Subclassification of the pleomorphic MLs according to the predominance of small, medium or large cells, demonstrated significant differences between these three subtypes. However, the great overlap of values between pleomorphic T-ML with medium and with large cells, seems to indicate that the subclassification of these two subtypes is less valid. (3) A wide range of values with overlap was observed in AILD-type ML, in pleomorphic with medium or large cells and in lymphoblastic T-ML: for these T-ML with variable survival courses, the Ki-67 area percentage, one parameter of proliferative activity, appears worth studying as a prognostic factor.     

Expression of nitric oxide synthases in primary ciliary dyskinesia

Nitric oxide is believed to play a central role in nonspecific defense of upper airways. Patients with primary ciliary dyskinesia have very low concentration of nasal nitric oxide, which may contribute to the chronic upper airway diseases encountered by these patients. The mechanisms underlying this drop of nasal nitric oxide in primary ciliary dyskinesia are still unknown. The goal of the present work was to study nitric oxide synthases expression in upper airway tissues from patients with primary ciliary dyskinesia. For this purpose, 5 patients with primary ciliary dyskinesia and 10 nonallergic age-matched patients without primary ciliary dyskinesia undergoing nasal polypectomy were included. Nasal nitric oxide concentration was measured before polypectomy, and nitric oxide synthase expression and function were studied in nasal polyps. The nasal nitric oxide in patients with primary ciliary dyskinesia was lower than that in patients without primary ciliary dyskinesia (13 [9-16] ppb versus 210 [167-254] ppb, P < .0001). Nitric oxide synthase 2 immunostaining was prominent at the apical part of the ciliated epithelial cells and was similar in both groups. Nitric oxide synthase 3 staining was restricted to endothelial cells in both groups. In addition, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was superimposable to nitric oxide synthases 2 and 3 immunostaining, suggesting a preserved NADPH-activity of nitric oxide synthase. We therefore conclude that the drop in nasal nitric oxide in patients with primary ciliary dyskinesia is not secondary to the loss of nitric oxide synthase expression.