Co-expression of Epstein-Barr virus latent membrane protein and vimentin in “aggressive” histological subtypes of Hodgkin's disease

Summary The presence of Epstein-Barr virus (EBV) genome in Hodgkin's and Reed-Sternberg (HRS) cells, as detected using in situ hybridization (ISH) with biotinylatedBamHI V probes, along with the expression of EBV-encoded latent membrane protein (LMP) and vimentin was examined in paraffin-embedded sections of 39 immunomorphologically characterized cases of Hodgkin's disease (HD). ISH demonstrated EBV in HRS cells in 15 of 39 cases, whereas LMP expression was detected in 11 of 39 cases, only in the presence of EBV genome detection. With the exception of 1 case, in which HRS cells expressed B-cell-associated antigens, the LMP-positive cases included specimens in which HRS cells were of non-B, non-T phenotype. LMP expression showed a stronger association with lymphocyte depletion (LD) (3/3) and mixed cellularity (MC) (6/11) than with lymphocyte predominance (0/5) or nodular sclerosis (2/20) subtypes. Vimentin expression on HRS cells was found in all the LMP-expressing cases and only in a fraction (13/28) of LMP-negative cases. This study supports the view that HD represents a heterogeneous group of diseases also in terms of EBV association, LMP expression being strongly related to the aggressive LD and MC histological subtypes. In light of the supposed interactions between vimentin and LMP, their coexpression on HRS cells, as detected in this study, provides further evidence for a significant role of EBV in the development of a proportion of HD cases.     

Inherited aplastic anemia with abnormal clones in bone marrow and increased endoreduplication in peripheral lymphocytes

Cytogenetic studies in peripheral blood and bone marrow cells from a female patient (aged 31 years) with inherited aplastic anemia and without other congenital anomalies are reported. Endoreduplication was increased in stimulated peripheral lymphocytes in several investigations. Chromosome breaks were shown to be near the control frequency, although chromatid exchange figures and dicentrics were present. Cytogenetic analysis was extended to the three children of our patient. Abnormal clones were detected in bone marrow preparations of our patient in all cytogenetic investigations. At the first examination, two of these clones were prevalent, with their karyotypes being 48,XX,+9,+16 and 46,XX,dup(1)(q24→q32),t(17;?)(p12–13;?). The prevailing karyotype after 2 years was 46,XX,t(17;?)(p12–13;?). Involvement of chromosomes #1 and #17 is discussed, taking into account data from the literature concerning several human neoplasias.     

ALK Expression Defines a Distinct Group of T/Null Lymphomas (“ALK Lymphomas”) with a Wide Morphological Spectrum

The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffin sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma “common” type (75%), “lymphohistiocytic” (10%), “small cell” (8.3%), “giant cell” (3.3%), and “Hodgkin’s like” (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin’s-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and “Hodgkin’s-like” features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype (“ALK lymphomas”), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.     

Large-scale variation among human and great ape genomes determined by array comparative genomic hybridization

Large-scale genomic rearrangements are a major force of evolutionary change and the ascertainment of such events between the human and great ape genomes is fundamental to a complete understanding of the genetic history and evolution of our species. Here, we present the results of an evolutionary analysis utilizing array comparative genomic hybridization (array CGH), measuring copy-number gains and losses among these species. Using an array of 2460 human bacterial artificial chromosomes (BACs) (12% of the genome), we identified a total of 63 sites of putative DNA copy-number variation between humans and the great apes (chimpanzee, bonobo, gorilla, and orangutan). Detailed molecular characterization of a subset of these sites confirmed rearrangements ranging from 40 to at least 175 kb in size. Surprisingly, the majority of variant sites differentiating great ape and human genomes were found within interstitial euchromatin. These data suggest that such large-scale events are not restricted solely to subtelomeric or pericentromeric regions, but also occur within genic regions. In addition, 5/9 of the verified variant sites localized to areas of intrachromosomal segmental duplication within the human genome. On the basis of the frequency of duplication in humans, this represents a 14-fold positional bias. In contrast to previous cytogenetic and comparative mapping studies, these results indicate extensive local repatterning of hominoid chromosomes in euchromatic regions through a duplication-driven mechanism of genome evolution.     

Synergistic effect of IFN-gamma and human cytomegalovirus protein UL40 in the HLA-E-dependent protection from NK cell-mediated cytotoxicity

Human CMV (HCMV) has evolved several strategies to evade the immune system of the infected host. Here, we investigated the role of the HCMV-encoded protein UL40 in the modulation of NK cell lysis. UL40 carries in its leader sequence a nonameric peptide similar to that found in many HLA class I molecules leader sequences. This peptide up-regulates the expression of HLA-E, the ligand for the NK cell inhibitory receptor CD94/NKG2A. The UL40-encoded HLA-E-binding peptide was present in all HCMV clinical (4636, 13B, 109B, 3C) and laboratory (AD169) strains analyzed. However, transfection of UL40 in different cell lines (293T, 721.221, K562) did not consistently confer protection from NK lysis (as measured using NKL and the newly generated NK line Nishi), despite a moderate up-regulation of HLA-E. Interestingly, combined transfection and treatment with IFN-gamma increased the inhibitory effect, via an HLA-E- and CD94/NKG2A-dependent mechanism. Although cells transfected with UL40 derived from either AD169 or 3C showed protection from NK cell lysis, infection of fibroblasts with the viruses resulted in a strong inhibition only with the clinical strain 3C. Our results suggest that UL40 and IFN-gamma-dependent up-regulation of HLA-E is only one possible mechanism to avoid NK cell recognition of HCMV infected cells.     

Selective depletion of the OKT 4+ 4B4+ subset in lymph nodes from HIV+ patients

We have used 4B4 and 2H4 monoclonal antibodies in conjunction with OKT 4 to quantify T cell subsets in lymph node suspensions from human immunodeficiency virus (HIV) positive subjects with lymphadenopathy syndrome. The data indicate that the reduced OKT 4:OKT 8 ratio was due to a depletion of the OKT 4+ 4B4+ subset. In contrast, there were no differences compared to reactive controls, considering the OKT8+ subpopulation. These alterations may be related to the immunological deficiency associated with HIV infection.     

BCL-6 protein expression in AIDS-related non-Hodgkin's lymphomas: inverse relationship with Epstein-Barr virus-encoded latent membrane protein-1 expression.

This study aimed at investigating the expression of the BCL-6 protein in acquired immune deficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (AIDS-NHLs) and at comparing the expression pattern with the presence of Epstein-Barr virus (EBV) infection of the tumor clone. A total of 34 AIDS-NHL biopsies, including 16 AIDS-related diffuse large-cell lymphomas (AIDS-DLCLs) and 18 AIDS-related small-noncleaved-cell lymphomas (AIDS-SNCCLs) as well as 7 AIDS-SNCCL cell lines were immunostained with a BCL-6-specific monoclonal antibody. Expression of BCL-6 protein was detected in 9 of 16 AIDS-DLCLs, 18 of 18 AIDS-SNCCLs, and 3 of 7 AIDS-SNCCL cell lines. Expression of BCL-6 among AIDS-NHLs occurred independently of the presence of molecular lesions of the bcl-6 gene. Notably, among EBV-positive AIDS-NHL biopsies and cell lines, expression of BCL-6 was mutually exclusive with the expression of EBV-encoded transforming antigen latent membrane protein-1. The mutual exclusion between BCL-6 and latent membrane protein-1 expression was further confirmed by in vitro superinfection experiments of an AIDS-SNCCL cell line originally devoid of EBV sequences. Overall, the frequent association between AIDS-NHLs and expression of BCL-6, a protein selectively expressed by germinal center B cells, suggests that these lymphomas may originate from the germinal center.