Leena Karenko

Germline Mutations in the Extracellular Domains of the 55 kDa TNF Receptor, TNFR1, Define a Family of Dominantly Inherited Autoinflammatory Syndromes

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.

Primary Cutaneous T-Cell Lymphomas Show a Deletion or Translocation Affecting NAV3, the Human UNC-53 Homologue

Multicolor fluorescent in situ hybridization (FISH) was used to identify acquired chromosomal aberrations in 12 patients with mycosis fungoides or Sézary syndrome, the most common forms of primary cutaneous T-cell lymphoma (CTCL). The most frequently affected chromosome was 12, which showed clonal deletions or translocations with a break point in 12q21 or 12q22 in five of seven consecutive Sézary syndrome patients and a clonal monosomy in the sixth patient. The break point of a balanced translocation t(12;18)(q21;q21.2), mapped in the minimal common region of two deletions, fine mapped to 12q2. By locus-specific FISH, the translocation disrupted one gene, NAV3 (POMFIL1), a human homologue of unc-53 in Caenorhabditis elegans. A missense mutation in the remaining NAV3 allele was found in one of six cases with a deletion or translocation. With locus-specific FISH, NAV3 deletions were found in the skin lesions of four of eight (50%) patients with early mycosis fungoides (stages IA-IIA) and in the skin or lymph node of 11 of 13 (85%) patients with advanced mycosis fungoides or Sézary syndrome. Preliminary functional studies with lentiviral small interfering RNA-based NAV3 silencing in Jurkat cells and in primary lymphocytes showed enhanced interleukin 2 expression (but not CD25 expression). Thus, NAV3 may contribute to the growth, differentiation, and apoptosis of CTCL cells as well as to the skewing from Th1-type to Th2-type phenotype during disease progression. NAV3, a novel putative haploinsufficient tumor suppressor gene, is disrupted in most cases of the commonest types of CTCL and may thus provide a new diagnostic tool.

Chromosomal abnormalities in relation to clinical disease in patients with cutaneous T-cell lymphoma: a 5-year follow-up study

Background  Patients with cutaneous T‐cell lymphoma (CTCL) show chromosomal aberrations in skin and blood lymphocytes.

Th1 Response and Cytotoxicity Genes Are Down-Regulated in Cutaneous T-Cell Lymphoma

Purpose: Increased production of Th2 cytokines characterizes Sezary syndrome, the leukemic form of cutaneous T-cell lymphomas (CTCL). To identify the molecular background and to study whether shared by the most common CTCL subtype, mycosis fungoides, we analyzed the gene expression profiles in both subtypes. Experimental Design: Freshly isolated cells from 30 samples, representing skin, blood, and enriched CD4+ cell populations of mycosis fungoides and Sezary syndrome, were analyzed with Affymetrix (Santa Clara, CA) oligonucleotide microarrays, quantitative PCR, or immunohistochemistry. The gene expression profiles were combined with findings of comparative genomic hybridization of the same samples to identify chromosomal changes affecting the aberrant gene expression. Results: We identified a set of Th1-specific genes [e.g., TBX21 (T-bet), NKG7, and SCYA5 (RANTES)] to be down-regulated in Sezary syndrome as well as in a proportion of mycosis fungoides samples. In both Sezary syndrome and mycosis fungoides blood samples, the S100P and LIR9 gene expression was up-regulated. In lesional skin, IL7R and CD52 were up-regulated. Integration of comparative genomic hybridization and transcriptomic data identified chromosome arms 1q, 3p, 3q, 4q, 12q, 16p, and 16q as likely targets for new CTCL-associated gene aberrations. Conclusions: Our findings revealed several new genes involved in CTCL pathogenesis and potential therapeutic targets. Down-regulation of a set of genes involved in Th1 polarization, including the major Th1-polarizing factor, TBX21, was for the first time associated with CTCL. In addition, a plausible explanation for the proliferative response of CTCL cells to locally produced interleukin-7 was revealed.

Allelic variants in genes associated with hereditary periodic fever syndromes as susceptibility factors for reactive systemic AA amyloidosis.

We investigated the hypothesis that low-penetrance mutations in genes (TNFRSF1A, MEFV and NALP3/CIAS1) associated with hereditary periodic fever syndromes (HPFs) might be risk factors for AA amyloidosis among patients with chronic inflammatory disorders, including rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), Crohns disease, undiagnosed recurrent fevers and HPFs themselves. Four of 67 patients with RA plus amyloidosis had MEFV variants compared with none of 34 RA patients without amyloid (P value=0.03). The E148Q variant of MEFV was present in two of the three patients with TNF receptor-associated periodic syndrome (TRAPS) complicated by amyloid in two separate multiplex TRAPS families containing 5 and 16 affected members respectively, and the single patient with Muckle–Wells syndrome who had amyloidosis was homozygous for this variant. The R92Q variant of TNFRSF1A was present in two of 61 JIA patients with amyloidosis, and none of 31 nonamyloidotic JIA patients. No HPF gene mutations were found in 130 healthy control subjects. Although allelic variants in HPFs genes are not major susceptibility factors for AA amyloidosis in chronic inflammatory disease, low-penetrance variants of MEFV and TNFRSF1A may have clinically significant proinflammatory effects.

Clinicopathological Characterization and Genomic Aberrations in Subcutaneous Panniculitis-Like T-Cell Lymphoma

Subcutaneous panniculitis-like T-cell lymphomas (SPTLs) represent a rare, difficult-to-diagnose, and poorly characterized subtype of cutaneous T-cell lymphomas (CTCLs) affecting younger people more than the other CTCL forms. We performed a thorough clinical, immunohistological, and molecular analysis of nine Finnish SPTL patients. Specifically, we performed single-cell comparative genomic hybridization (CGH) from laser microdissected, morphologically malignant SPTL cells, as well as loss of heterozygosity (LOH) and fluorescence in situ hybridization (FISH) analysis for the NAV3 (neuron navigator 3) gene. CGH revealed large numbers of DNA copy number changes, the most common of which were losses of chromosomes 1pter, 2pter, 10qter, 11qter, 12qter, 16, 19, 20, and 22 and gains of chromosomes 2q and 4q. Some of the DNA copy number aberrations in SPTL, such as loss of 10q, 17p, and chromosome 19, overlap with those characteristic of common forms of CTCL (mycosis fungoides (MF) and Sezary syndrome (SS)), whereas 5q and 13q gains characterize SPTL. Allelic NAV3 aberrations (LOH or deletion by FISH), previously found in MF and SS, were identified in 44% of the SPTL samples. This study demonstrates that SPTL is also moleculocytogenetically a uniform entity of CTCL and supports the current World Health Organization-European Organization for Research and Treatment of Cancer (WHO-EORTC) classification defining SPTL as a subgroup of its own.

Proapoptotic and antiapoptotic markers in cutaneous T-cell lymphoma skin infiltrates and lymphomatoid papulosis

Background In cutaneous T‐cell lymphoma (CTCL) lesions, both reactive T cells and malignant T cells intermingle. The disease progression is mostly slow. Recent evidence suggests that even if clinical remission is reached, malignant cells persist and a relapse follows sooner or later. To what extent tumour cell apoptosis occurs in the skin lesions either due to the reactive T cells or to therapeutic efforts is not known.

Expression of Human Endogenous Retrovirus-W Including Syncytin-1 in Cutaneous T-Cell Lymphoma

The pathomechanism of mycosis fungoides (MF), the most common type of primary cutaneous T-cell lymphomas (CTCLs) and a malignancy of non-recirculating, skin-resident T-cells, is unknown albeit underlying viral infections have been sought for. Human endogenous retroviruses (HERVs) are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancers. We explored the transcriptional activity of HERV sequences in a total of 34 samples comprising MF and psoriasis skin lesions, as well as corresponding non-malignant skin using a retrovirus-specific microarray and quantitative RT-PCR. To identify active HERV-W loci, we cloned the HERV-W specific RT-PCR products, sequenced the cDNA clones and assigned the sequences to HERV-W loci. Finally, we used immunohistochemistry on MF patient and non-malignant inflammatory skin samples to confirm specific HERV-encoded protein expression. Firstly, a distinct, skin-specific transcription profile consisting of five constitutively active HERV groups was established. Although individual variability was common, HERV-W showed significantly increased transcription in MF lesions compared to clinically intact skin from the same patient. Predominantly transcribed HERV-W loci were found to be located in chromosomes 6q21 and 7q21.2, chromosomal regions typically altered in CTCL. Surprisingly, we also found the expression of 7q21.2/ERVWE1-encoded Syncytin-1 (Env) protein in MF biopsies and expression of Syncytin-1 was seen in malignant lymphocytes, especially in the epidermotropic ones, in 15 of 30 cases studied. Most importantly, no Syncytin-1 expression was detected in inflammatory dermatosis (Lichen ruber planus) with skin-homing, non-malignant T lymphocytes. The expression of ERVWE1 mRNA was further confirmed in 3/7 MF lesions analyzed. Our observations strengthen the association between activated HERVs and cancer. The study offers a new perspective into the pathogenesis of CTCL since we demonstrate that differences in HERV-W transcription levels between lesional MF and non-malignant skin are significant, and that ERVWE1-encoded Syncytin-1 is expressed in MF lymphoma cells.

Molecular cytogenetics in the study of cutaneous T-cell lymphomas (CTCL)

The etiology of primary cutaneous T-cell lymphomas (CTCL) has been poorly understood. CTCL patients show a large variety of non-clonal and clonal chromosome aberrations, but no specific aberration has been found until recently. This review describes cytogenetic and molecular cytogenetic findings and their relevance to diagnostics and etiology in two of the most common forms of CTCL, mycosis fungoides (MF) and the leukemic CTCL, Sézary syndrome (SS).

Low TNF-induced NF-κB and p38 phosphorylation levels in leucocytes in tumour necrosis factor receptor-associated periodic syndrome

OBJECTIVE TNF receptor-associated periodic syndrome (TRAPS) is a systemic autoinflammatory disorder caused by mutations in the type 1 TNF receptor (TNFRSF1A) gene. Because the pathomechanism of TRAPS may involve aberrant TNF-mediated intracellular signalling, we examined phosphorylation levels of nuclear factor kappaB (NF-kappaB) and p38 in response to TNF in 10 patients with three different TNFRSF1A mutations (C73R, C88Y and F112I). METHODS Phosphorylation levels of NF-kappaB p65 and p38 were determined in fresh leucocytes stimulated with TNF (0-100 ng/ml) for 2.5-20 min and permeabilized for phospho-specific antibodies in a whole blood flow cytometry assay. As control agonists, we used bacterial lipopolysaccharide (LPS) and IFN-gamma, the latter mediating phosphorylation of the signal transducer and activator of transcription 1. Areas under curve values for dose-response and time course of NF-kappaB and p38 phosphorylation were calculated for the comparison of patients and reference subjects. RESULTS NF-kappaB and p38 phosphorylation levels of monocytes, lymphocytes and neutrophils stimulated with TNF were significantly lower in TRAPS patients than in reference subjects. Phosphorylation levels induced by LPS, or by IFN-gamma, in patient and reference samples were comparable, indicating that the defect was confined to TNF-mediated signalling. CONCLUSIONS In the three families studied, TRAPS was associated with low TNF-mediated signalling in leucocytes. This deficiency of the innate immune system may result in the activation of as yet unidentified compensatory regulatory mechanisms yielding the hyperinflammatory phenotype of TRAPS.