Human herpesvirus 6 positive Reed-Sternberg cells in nodular sclerosis Hodgkin lymphoma.

Classical Hodgkin lymphoma (HL) exhibits a bi-modal age distribution that suggests an infectious aetiology. However, most cases of nodular sclerosis HL (NSHL) are Epstein-Barr virus (EBV) negative (60-90%). Previous studies regarding human herpesvirus 6 (HHV-6) positivity of HL have led to conflicting results. In order to clarify this situation, we examined NSHL biopsies for the presence and distribution of HHV-6 by immunohistochemistry (IHC), polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH). PCR identified HHV-6 DNA in 86% of NSHL cases. As HHV-6 DNA was also identified in most cases of reactive lymphoid hyperplasia, we sought to localize the virus to specific cells by IHC, which detected HHV-6 in Reed-Sternberg (RS) cells of nearly half (48%) of NSHL cases. Dual CD30/HHV-6 immunostaining confirmed HHV-6 immunoreactivity in CD30+ RS cells, and HHV-6 PCR positivity was confirmed in laser capture microdissection-isolated CD30+ RS cells. FISH demonstrated multiple copies of HHV-6 genome in scattered cells. In contrast, EBV+ RS cells were identified in only 24% of the cases. HHV-6+ cases trended toward a younger age than EBV+ cases. These results conclusively demonstrate that RS cells in many cases of NSHL are HHV-6 positive, and suggest that HHV-6 may play a role in NSHL pathogenesis, particularly in younger patients with EBV-negative disease.

p120 nucleolar-proliferating antigen is a direct target of G-CSF signaling during myeloid differentiation.

Granulocyte-colony stimulating factor (G-CSF) is an essential cytokine, which contributes to proliferation and differentiation of granulocyte precursor cells in the bone marrow. Despite recent progress in understanding G-CSF signaling events, the mechanisms that underlie the distinct spectrum of biological functions attributed to G-CSF-mediated gene expression remain unclear. Previous studies have identified a number of genes, which are up-regulated in G-CSF-stimulated myeloid precursor cells. In this study, we sought to identify additional target genes of G-CSF-mediated proliferation and/or differentiation. cDNA representational difference analysis was used with the 32Dcl3 cell line as a model system to isolate genes, which are up-regulated in an immediate-early manner upon G-CSF stimualtion. We isolated p120 nucleolar-proliferation antigen (NOL1), a highly conserved, nucleolar-specific, RNA-binding protein of unknown function, and confirmed its expression by Northern blot analysis in 4-h, G-CSF-induced 32Dcl3 cells. Isolation of a mouse p120 genomic clone revealed the presence of a signal tranducer and activator of transcription (STAT)-binding site in the first intron of the gene. We demonstrate the importance of STAT3 and STAT5 in mediating the G-CSF response with respect to p120 expression by transient transfection analysis, oligonucleotide pull-down assays, and the loss of p120 expression in the bone marrow of mice lacking normal STAT3 signaling. In addition, overexpression of p120 in G-CSF-induced 32D cells revealed normal, morphologic maturation and growth characteristics but loss of lactoferrin expression, a marker of normal neutrophil maturation, suggesting that inappropriate expression of the p120 gene can result in aberrant neutrophil maturation.

Recurrent genetic defects in classical Hodgkin lymphoma cell lines.

Genetic analysis of classical Hodgkin lymphoma (cHL) has been hampered by the paucity of Hodgkin cells in biopsies and their poor growth in vitro. However, a wealth of information has been obtained from cHL cell lines. Here we report results of whole-exome sequencing and karyotypic analysis of five cHL cell lines. Four genes with potentially pathogenic single nucleotide variants (SNV) were detected in three cell lines. SNV were also detected in seventeen HL-related genes and three mitosis-related genes. Copy number variants were detected in four HL-related genes in all five cell lines. Given the high degree of aneuploidy in HL, mitosis-related genes were screened for defects. One mitotic gene (NCAPD2) was amplified in all five HL cell lines, and two genes (FAM190A, PLK4) were amplified in four cell lines. These results suggest that genomic instability of HL may be due to defects in genes involved in chromosome duplication and segregation.

CASK deletion in intestinal epithelia causes mislocalization of LIN7C and the DLG1/Scrib polarity complex without affecting cell polarity.

CASK is the mammalian ortholog of LIN2, a component of the LIN2/7/10 protein complex that targets epidermal growth factor receptor (EGFR) to basolateral membranes in Caenorhabditis elegans. A member of the MAGUK family of scaffolding proteins, CASK resides at basolateral membranes in polarized epithelia. Its interaction with LIN7 is evolutionarily conserved. In addition, CASK forms a complex with another MAGUK, the DLG1 tumor suppressor. Although complete knockout of CASK is lethal, the gene is X-linked, enabling us to generate heterozygous female adults that are mosaic for its expression. We also generated intestine-specific CASK knockout mice. Immunofluorescence analysis revealed that in intestine, CASK is not required for epithelial polarity or differentiation but is necessary for the basolateral localization of DLG1 and LIN7C. However, the subcellular distributions of DLG1 and LIN7C are independent of CASK in the stomach. Moreover, CASK and LIN7C show normal localization in dlg1(-/-) intestine. Despite the disappearance of basolateral LIN7C in CASK-deficient intestinal crypts, this epithelium retains normal localization of LIN7A/B, EGFR and ErbB-2. Finally, crypt-to-villus migration rates are unchanged in CASK-deficient intestinal epithelium. Thus, CASK expression and the appropriate localization of DLG1 are not essential for either epithelial polarity or intestinal homeostasis in vivo.