The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes

International audienceBackground: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. Materials and Methods: Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. Results: Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30−/CD15− cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival (p < 10−3), event-free survival (p < 10−4), and freedom from progression (p < 10−3) and the presence of an ALT profile in lymph nodes of EBV+ patients. Conclusion: The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients

Delphinella Shoot Blight on Abies lasiocarpa Provenances in Norway

Delphinella shoot blight (Delphinella abietis) attacks true firs (Abies spp.) in Europe and North America. Especially subalpine fir (A. lasiocarpa), one of the main Christmas tree species in Norway, is prone to the disease. The fungus kills current year needles, and in severe cases entire shoots. Dead needles become covered with black fruiting bodies, both pycnidia and pseudothecia. Delphinella shoot blight has mainly been a problem in humid, coastal regions in the northwestern part of Southern Norway, but, probably due to higher precipitation in inland regions during recent years, heavy attacks were found in 2011 in a field trial with 76 provenances of subalpine fir in Southeastern Norway. However, the amount of precipitation seemed less important once the disease had established in the field. Significant differences in susceptibility between provenances were observed. In general, the more bluish the foliage was, the healthier the trees appeared. The analysis of provenance means indicated that, at least for the southern range, the disease ratings were correlated with foliage color. This study also includes isolation, identification, a pathogenicity test, a seed test and electron microscopy of the wax layer on the needles. The fungus was identified based on the morphology of spores and by sequencing the Internal Transcribed Spacer (ITS) regions of the ribosomal DNA. Koch’s postulates were fulfilled. The fungus was found present on newly harvested seeds and may therefore spread via international seed trade. When comparing the wax layers on green and blue needles, those of the latter were significantly thicker, a factor that may be involved in disease resistance

Hymenosetin, a 3-decalinoyltetramic acid antibiotic from cultures of the ash dieback pathogen, Hymenoscyphus pseudoalbidus.

A 3-decalinoyltetramic acid, for which the trivial name hymenosetin is proposed, was isolated from crude extracts of a virulent strain of the ash dieback pathogen, Hymenoscyphus pseudoalbidus (="Chalara fraxinea"). This compound was produced only under certain culture conditions in submerged cultures of the fungus. Its planar structure was determined by NMR spectroscopy and by mass spectrometry. The absolute stereochemistry was assigned by CD spectroscopy and HETLOC data. Hymenosetin exhibited broad spectrum antibacterial and antifungal activities (including strong inhibition of MRSA), as well as moderate cytotoxic effects. So far, the metabolite proved inactive in assays for evaluation of phytotoxicity, whereas viridiol, another secondary metabolite known from H. pseudoalbidus, was regarded as phytotoxic principle of the pathogen against its host, Fraxinus excelsior. Further studies will show whether hymenosetin constitutes a defence metabolite that is produced by the pathogenic fungus to combat other microbes and fungi in the natural environment

Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability †

Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL

Erratum: Cuceu, C., et al. Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability. <i>Cancers</i> 2018, <i>10</i>, 233

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Data from: Unravelling hybridization in Phytophthora using phylogenomics and genome size estimation

The genus Phytophthora comprises many economically and ecologically important plant pathogens. Hybrid species have previously been identified in at least six of the 12 phylogenetic clades. These hybrids can potentially infect a wider host range and display enhanced vigour compared to their progenitors. Phytophthora hybrids therefore pose a serious threat to agriculture as well as to natural ecosystems. Early and correct identification of hybrids is therefore essential for adequate plant protection but this is hampered by the limitations of morphological and traditional molecular methods. Identification of hybrids is also important in evolutionary studies as the positioning of hybrids in a phylogenetic tree can lead to suboptimal topologies. To improve the identification of hybrids we have combined genotyping-by-sequencing (GBS) and genome size estimation on a genus-wide collection of 614 Phytophthora isolates. Analyses based on locus- and allele counts and especially on the combination of species-specific loci and genome size estimations allowed us to confirm and characterize 27 previously described hybrid species and discover 16 new hybrid species. Our method was also valuable for species identification at an unprecedented resolution and further allowed correct naming of misidentified isolates. We used both a concatenation- and a coalescent-based phylogenomic method to construct a reliable phylogeny using the GBS data of 140 non-hybrid Phytophthora isolates. Hybrid species were subsequently connected to their progenitors in this phylogenetic tree. In this study we demonstrate the application of two validated techniques (GBS and flow cytometry) for relatively low cost but high resolution identification of hybrids and their phylogenetic relations

Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30&#8722;/CD15&#8722; cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (&#8722;/&#8722;)(NSG) mice. Using cell sorting, we demonstrate that CD30&#8722;/CD15&#8722; subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30&#8722;/CD15&#8722; cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 103 cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30&#8722;/CD15&#8722; cells exhibiting high telomerase activity and telomere dysfunction